05 Proexp PDF

Novagen
Protein Expression
Related Products & Applications
Upstream Starting Target Application Downstream
1 Application Material Application
Novagen Tools in this Chapter

2
Protein Expression
3 Prokaryotic Expression See this chapter
PCR
pETBlue™ System
See PCR, Chapter 1 pET System Protein Purification
4 Host Strains and Competent Cells See Protein Purification,
Chapter 9
Insect Cell Expression

5 Target Protein BacVector ® Baculovirus System
Protein
Structural Studies
Cloning Coding Transient and Stable Expression See Proteases in
See Cloning, Sequence Protein & Gene Analysis,
Chapter 2
Plasmids Chapter 7
6 ATG
GCT
AAA
GCT
GAA
GCT
ACC
AAA
Insect Cells, Media and Reagents
TTC GAA CGC CAG
CAC ATG GAC AGC
Gel Electrophoresis
See Molecular Size
Mammalian Expression Markers, Chapter 11
7 Transient Expression Vectors
Phage Display Stable Expression Vectors
See Phage Display,
Reporter Assay
Transfection and Selection Tools See Protein & Gene
Chapter 4
8 Analysis, Chapter 7
Multisystem Expression Fusion Tag Assay

pTriEx™ Multisystem Expression and Detection
9 Vectors See Protein & Gene
Analysis, Chapter 7
DNA Purification
See Nucleic Acid
Purification,
10 Chapter 10 Immunoscreening
See Tissues & Libraries,
Chapter 8
11
Appendices
Indices
82 Novagen 2002–2003 Catalog

Novagen
Protein Expression
Table of Contents
1
Prokaryotic Expression Insect Cell Expression
pETBlue™ and pET System Insect Cell Expression Overview . . . .116
BacVector® System . . . . . . . . . . . . .117
Overview . . . . . . . . . . . . . . . . . . . . .84
pETBlue System . . . . . . . . . . . . . . . . .86 BacVector Transfection Kits . . . . . . .118
2
pETBlue PCR Cloning Kits . . . . . . . . . .87 Baculovirus Transfer Plasmids . . . . .119
pET System Tutorial . . . . . . . . . . . . . .88 pBAC™ E. coli LIC Vector Kits . . . . .121
pET Vector DNA . . . . . . . . . . . . . . . . .93 Insect Cell Transient and Stable
pET Expression Systems . . . . . . . . . . .94 Expression Plasmids . . . . . . . . . . .122 3
Fusion Tag Affinity Purification pBAC, pAcPIE1, and pIE1 Plasmid
Products . . . . . . . . . . . . . . . . . . . . .96 Primers . . . . . . . . . . . . . . . . . . . . .123
pET Expression Systems FastPlax™ Titer Kit . . . . . . . . . . . . . .123
3, 9, 11, 17b, and 17xb . . . . . . . . . .97 Sf9 Insect Cells . . . . . . . . . . . . . . . .124
pET Expression Systems BacVector Insect Cell Medium . . . . .124 4
14b, 15b, 16b, and 19b . . . . . . . . . .98 TriEx Sf9 Cells . . . . . . . . . . . . . . . . .124
pET-21(+), 23(+), and 24(+) TriEx Insect Cell Medium . . . . . . . . .124
Transcription Vectors . . . . . . . . . . . .98 Eufectin™ Transfection Reagent . . .125
pET Expression Systems BacPlaque™ Agarose . . . . . . . . . . . .125
12, 20b, 22b, 25b, 26b, and 27b . . .99 X-Gluc Solution . . . . . . . . . . . . . . . . .125 5
pET Expression Systems
21, 23, and 24 . . . . . . . . . . . . . . .100 Mammalian Expression
pET Expression Systems Mammalian Expression
28, 29, and 30 . . . . . . . . . . . . . . .100 Introduction . . . . . . . . . . . . . . . . . .126
pET Peptide Expression pTriEx Transient Expression 6
System 31b . . . . . . . . . . . . . . . . . .101 Vectors . . . . . . . . . . . . . . . . . . . . . .126
pET Trx Fusion System 32 . . . . . . . .102 pTriEx-4 Ek/LIC Vector Kit . . . . . . . . .127
pET Expression System 33b . . . . . . .103 pTriEx Stable Expression Vectors . . .128
PKAce™ Kit . . . . . . . . . . . . . . . . . . .103
pET CBD Fusion Systems
GeneJuice™ Transfection
Reagent . . . . . . . . . . . . . . . . . . . . .130
7
34b–38b . . . . . . . . . . . . . . . . . . . .104 Antibiotics . . . . . . . . . . . . . . . . . . . .131
pET Dsb Fusion Systems
39b and 40b . . . . . . . . . . . . . . . . .105 Multisystem Expression
pET GST Fusion Systems pTriEx Multisystem Expression 8
41 and 42 . . . . . . . . . . . . . . . . . . .106 Vectors . . . . . . . . . . . . . . . . . . . . . .132
pET NusA Fusion System 43.1 . . . . .107
pET LIC Vector Kits . . . . . . . . . . . . . .108
pET Host Strain Glycerol Stocks . . . .110
100 mM IPTG Solution . . . . . . . . . . .111 9
pET Host Strain Competent Cells . . .112
pET Competent Cell Sets . . . . . . . . .113
pETBlue and pTriEx™ Host Strain
Competent Cells . . . . . . . . . . . . . . .113
Singles™ Competent Cells . . . . . . . .114 10
HT96™ Competent Cells . . . . . . . . .114
Bacteriophage CE6 . . . . . . . . . . . . . .115
lDE3 Lysogenization Kit . . . . . . . . . .115
11
Appendices
Indices
Novagen 2002–2003 Catalog 83

Novagen
Protein Expression
Prokaryotic Expression
1 pETBlue™ and pET System Overview

A new generation, a trusted standard
Protein Expression in Bacteria porate the same T7-based expression controls as pET System: The Gold Standard
the pETBlue vectors, plus they extend the use of for Protein Expression in E. coli
For many researchers around the world,
a single construct to express target proteins in
Novagen’s pET System is the overwhelming In pET vectors, target genes are cloned
2 choice for protein expression in E. coli. A prima-
insect and mammalian hosts. Please see
Multisystem Expression later in this chapter for
under control of strong bacteriophage T7 tran-
ry reason for the success of this system is that scription and translation signals, and expression
more information on the pTriEx vectors.
target genes are cloned under the control of the is induced by providing a source of T7 RNA poly-
T7 promoter, which is not recognized by E. coli pETBlue System: The New Generation of T7 merase in the host cell. Novagen’s pET System
RNA polymerase. Therefore, virtually no expres- Expression Vectors has continuously expanded to offer new tech-
3 sion occurs until a source of T7 RNA polymerase

is provided. Genes cloned in pET vectors are vir-
The pETBlue vectors represent a fundamen-
nologies and options for expression, and
includes over 36 pET vector types, 13 different
tally new type of expression vector, combining
tually “off” and cannot cause plasmid instability host strains and many other companion products
the most desirable features of popular cloning
due to the production of proteins potentially designed for efficient detection and purification
vectors with the full power of T7-driven protein
toxic to the host cell. of target proteins.
expression. Blue/white visual screening of
Once established, plasmids are transferred Features
4 into expression hosts containing a chromosomal
copy of the T7 RNA polymerase gene under
recombinants is enabled by insertion of target
genes into the lacZ a-peptide coding region. • The number-one cited system for prokaryotic
Expression is made possible by T7 transcription protein expression
lacUV5 control, and expression is induced by the
and translation signals. As with standard pET • Lowest basal expression levels of any E. coli
addition of IPTG. Alternatively, T7 RNA poly-
vectors, target proteins are produced by transfer expression system
merase can be provided by infection of the origi-
into a lDE3 lysogen followed by IPTG induction • “Tunable” control for modulating expression
5 nal cloning host with lCE6. Many genes that
have been difficult to establish in E. coli promot-
er-based systems (e.g., tac, lac, trc, pL ) have
or by infection of the original host with lCE6.
Features
levels if desired
• Widest variety of fusion tags and
been stably cloned and expressed in the pET • Blue/white screening for easy cloning configurations of any expression system
System. T7 RNA polymerase is so selective and • High-copy number for high plasmid DNA • Specialized vectors and hosts for production
active that almost all of the cell’s resources are yields of soluble proteins, disulfide bond formation,
6 converted to target gene expression. The desired
product can comprise more than 50% of the total
• Available as AccepTor™ Vector or Perfectly protein export, peptide production, etc.
Blunt® Vectors for rapid PCR cloning • Many vectors available as LIC Vector Kits for
cell protein a few hours after induction. Many
• No basal expression of target genes; rapid, directional cloning of PCR products
other advantages of Novagen’s pET System are
discussed in the following sections. eliminates plasmid instability associated with • Most host strains available as competent
Other advances in T7-driven expression tech- toxic gene products cells, ready for transformation
7 nology are represented by the pETBlue™ System

and pTriEx™ vectors. The pETBlue vectors
• Same expression levels as classic pET
vectors
continued on next page
incorporate all of the advantages of pET vectors • “Tunable” control of expression levels with
for expression, while also increasing ease of use Tuner™(DE3)pLacI and
for target gene cloning and manipulation of plas- Rosetta™(DE3)pLacI host strains
mid DNA. The pTriEx multisystem vectors incor-
8
insert insert
Blue ss ion
on /Wh pre
ss i ite Ex T7
pre
T7
9 Ex
RBS Sc
re
en
RBS
MCS lacZ M CS
lacZ lacO
T7 t e
in
tet r m.
g
O pro .
lac m. T7 rom
p
.
m
pro7
10
T
T7
pET System
t
erm
O
lac
pETBlue™ System
11
Appendices
Indices
Orders: www.novagen.com Shipping and Storage Information: begins on page 280

Technical Support: [email protected] Ordering and Bulk Order Information: see page 10
Novagen
Protein Expression
pETBlue™ and pET System Overview continued

1
pETBlue Vectors
Common features of the pETBlue Vectors: Fusion Tags Protease Special
2
• Tightly controlled, T7 dual lacO promoter Vector N-terminal C-terminal Cleavage Sites Features/Applications
• Ampicillin resistance marker
pETBlue-1 none none none Cloning/expression of target genes having their
• High-copy plasmid origin of replication own ATG start codons.
•
•
f1 origin of replication
Blue/white screening
pETBlue-2 none HSV•Tag®
His•Tag®
none Cloning/expression from vector-encoded ATG
(like most pET vectors); optional C-terminal tags. 3
• Available as AccepTor™ Vector (pETBlue-1)
or Perfectly Blunt® Vectors for cloning PCR
products
pET Vectors
4
Common features of the pET Vectors: These vectors are available as linearized LIC • pET-34b(+)
• pBR322 plasmid origin of replication vectors for ligation-independent cloning of PCR • pET-35b(+)
• f1 origin of replication [in (+) vectors]
Most pET vectors listed here provide ATG
products:
• pET-30 Ek/LIC
•
•
pET-36b(+)
pET-37b(+)
5
start codons and ATG cloning sites (Nde I or • pET-30 Xa/LIC • pET-41 Ek/LIC
Nco I). pET-21(+), pET-23(+) and pET-24(+) do • pET-32 Ek/LIC • pET-43.1 Ek/LIC
not provide RBS or ATG start codons. • pET-32 Xa/LIC
Fusion Tags Protease

6
Vector Promoter Selection N-terminal C-terminal Cleavage Sites Special Features/Applications
pET-3a–d T7 Ap T7•Tag® none none Basic pET vectors, offer single BamH I cloning site in 3 frames,
pET-9a–d T7 Kan T7•Tag none none except for pET-17b and pET-17xb, which have multiple cloning
pET-11a–d T7lac Ap T7•Tag none none sites in one frame. N-terminal T7•Tag epitope.
pET-17b T7 Ap T7•Tag none none
pET-17xb
pET-12a–c
T7
T7
Ap
Ap
T7•Tag
ompT
none
none
none
SP Produce signal sequence fusions to facilitate export of target
7
pET-20b(+) T7 Ap pelB His•Tag® SP proteins to the periplasm. Signal sequence cleaved by signal
pET-22b(+) T7lac Ap pelB His•Tag SP peptidase (SP) concomitant with export.
pET-25b(+) T7lac Ap pelB HSV•Tag®/His•Tag SP
pET-26b(+) T7lac Kan pelB His•Tag SP
pET-27b(+) T7lac Kan pelB HSV•Tag/His•Tag SP
pET-14b
pET-15b
T7
T7lac
Ap
Ap
His•Tag
His•Tag
none
none
Tb
Tb
Basic cleavable N-terminal His•Tag® fusion vectors, single
frame with 3 cloning sites.
8
pET-16b T7lac Ap His•Tag none Xa
pET-19b T7lac Ap His•Tag none Xa
pET-21a–d(+) T7lac Ap T7•Tag His•Tag none Combination of N-terminal T7•Tag® epitope and C-terminal
pET-23a–d(+) T7 Ap T7•Tag His•Tag none His•Tag sequence. Multiple cloning sites in 3 frames.
pET-24a–d(+) T7lac Kan T7•Tag His•Tag none
pET-28a–c(+)
pET-29a–c(+)
T7lac
T7lac
Kan
Kan
His•Tag/T7•Tag
S•Tag
His•Tag
His•Tag
Tb
Tb
Cleavable N-terminal His•Tag/T7•Tag or S•Tag™/His•Tag,
and C-terminal His•Tag. Multiple cloning sites in 3 frames.
9
pET-30a–c(+) T7lac Kan His•Tag/S•Tag His•Tag Tb, Ek Ek/LIC and Xa/LIC versions for PCR cloning (as LIC kits),
pET-30 Ek/LIC T7lac Kan His•Tag/S•Tag His•Tag Tb, Ek removal of all N-terminal tag aa.
pET-30 Xa/LIC T7lac Kan His•Tag/S•Tag His•Tag Tb, Xa
pET-31b(+) T7lac Ap KSI His•Tag none KSI fusions for high expression levels in inclusion bodies. Ideal
for peptide production. AlwN I cut vector available.
pET-32a–c(+) T7lac Ap Trx•Tag/His•Tag/S•Tag His•Tag Tb, Ek Cleavable Trx•Tag™ increases solubility of target proteins. 10
pET-32 Ek/LIC T7lac Ap Trx•Tag/His•Tag/S•Tag His•Tag Tb, Ek Multiple cloning sites in 3 frames. Ek/LIC and Xa/LIC versions for
pET-32 Xa/LIC T7lac Ap Trx•Tag/His•Tag/S•Tag His•Tag Tb, Xa PCR cloning (as LIC kits) and removal of all N-terminal tag aa.
pET-33b(+) T7lac Kan His•Tag/PKA/ His•Tag Tb Kinase site for in vitro 32P labeling of target proteins with
T7•Tag PKAce™ Kit.
pET-34b(+) T7lac Kan CBDclos•Tag/S•Tag His•Tag Tb, Ek Cleavable CBD fusion sequences. CBDcenA and CBDcex constructs
pET-35b(+)
pET-36b(+)
T7lac
T7lac
Kan
Kan
CBDclos•Tag/S•Tag
CBDcenA•Tag/S•Tag
His•Tag
His•Tag
Tb, Xa
SP, Tb, Ek
provide export signals. Ek/LIC and Xa/LIC versions for PCR
cloning (as LIC kits) and removal of all N-terminal tag aa. 11
pET-37b(+) T7lac Kan CBDcenA•Tag/S•Tag His•Tag SP, Tb, Xa
pET-38b(+) T7lac Kan CBDcex signal CBDcex•Tag/His•Tag SP, Tb
pET-39b(+) T7lac Kan DsbA•Tag/His•Tag/S•Tag His•Tag SP, Tb, Ek Cleavable Dsb sequences for export and proper folding in
Appendices
pET-40b(+) T7lac Kan DsbA•Tag/His•Tag/S•Tag His•Tag SP, Tb, Ek periplasm.

Indices
pET-41b(+) T7lac Kan GST•Tag/His•Tag/S•Tag His•Tag Tb, Ek Cleavable N-terminal GST•Tag™/His•Tag/ S•Tag, and C-
pET-42b(+) T7lac Kan GST•Tag/His•Tag/S•Tag His•Tag Tb, Xa terminal His•Tag.
pET-43.1a–c(+) T7lac Ap Nus•Tag/His•Tag/S•Tag HSV•Tag/His•Tag Tb, Ek Cleavable Nus•Tag™ sequence increases solubility of target
proteins. Multiple cloning sites in 3 frames.

Novagen 2002–2003 Catalog
85
Novagen
Protein Expression
1 pETBlue™ System Product Cat. No.
Tightly controlled T7-driven expression plus blue/white screening and high plasmid yield pETBlue™-1 System 70673-3
pETBlue-2 System 70674-3
The novel pETBlue™ System combines the are also compatible with pETBlue constructs.
Components:
visual identification of recombinants and high These hosts carry a chromosomal copy of the T7
• 20 µg pETBlue-1 DNA or
plasmid copy number, found in popular RNA polymerase gene under lacUV5 control, and pETBlue-2 DNA
2 blue/white screening vectors, with the tightly
controlled high yield protein expression
supply sufficient lac repressor via the compati-
ble pLacI plasmid to ensure low level uninduced
• 0.2 ml
• 0.2 ml
NovaBlue Competent Cells
Tuner™(DE3)pLacI Competent
obtained with pET vectors. Blue/white screening expression. Cells
is achieved using a weak constitutive E. coli pro- • 2 × 2 ml SOC Medium
pETBlue-1 • 2 ng Test Plasmid
moter (tet) to drive expression of the lacZ a-pep-
tide, whereas expression of target genes is driv- pETBlue-1 facilitates the expression of • 500 pmol pETBlueUP Primer
3 en by a T7lac promoter in the opposite orienta- unfused, native protein from inserts that encode • 500 pmol pETBlueDOWN Primer
tion. Insertion of target sequences into the multi- an open reading frame at the 5'-end. The EcoR V Available separately:
ple cloning site (MCS) disrupts expression of the cloning site (GATATC) in this vector is optimally Product Size Cat. No.
lacZ a-peptide and produces a white colony phe- positioned relative to the strong T7 gene 10 ribo-
pETBlue-1 DNA* 20 µg 70608-3
notype in strain NovaBlue when plated in the some binding site (RBS) as shown below. Inserts
pETBlue-2 DNA* 20 µg 70609-3
4 presence of X-gal. Colonies derived from the
unmodified vector turn blue. Because T7-driven
that either possess an ATG start codon or an
appropriately positioned G-nucleotide at the 5'- pETBlueUP Primer 500 pmol 70604-3
protein expression requires inserts to be cloned end will create an optimal E. coli translation pETBlueDOWN
in an antisense orientation relative to the tet pro- initiation site. Primer 500 pmol 70603-3
moter, basal expression of target sequences is
pETBlue-2 NovaBlue 0.4 ml 69825-3
virtually absent. The high-copy number pUC ori- Competent Cells 1 ml 69825-4
5 gin of replication present on the pETBlue plas-
mids greatly increases plasmid yields relative to
pETBlue-2 provides a vector-encoded ATG
start codon and variety of downstream cloning
Tuner(DE3)pLacI
Competent Cells
0.4 ml
1 ml
70625-3
70625-4
the pET vectors and provides an advantage for sites. In-frame cloning of any insert is facilitated
* Expression hosts containing pLacI are required with pETBlue
sequencing, mutagenesis and other plasmid by the presence of two sets of three overlapping
vectors
manipulations. blunt cutting sites at both the 5'- and 3'-ends of
Target genes in pETBlue vectors can be the multiple cloning sites (see diagram below
6 expressed at high levels, provided that the and sequence in Appendix). The blunt end creat- Additional Information Available
inserted sequences are in the sense orientation ed by each of these enzymes terminates in a dif- pET System Manual TB055
relative to the T7lac promoter, and meet the ferent position of the codon triplet in the vector- pETBlue System Protocol TB249
translation requirements defined by each vector defined reading frame. Therefore, when inserted Vector Cloning Region Sequences Appendix
Vector maps and sequences www.novagen.com
(see below). Protein expression is accomplished in the appropriate orientation, any insert can be Patent and Licensing Information p. 278
7 in one of two ways; by infection with lCE6 (a
phage that expresses T7 RNA polymerase under
cloned in-frame by cutting the vector with the
appropriate combination of blunt cutting
control of the l pL promoter), or by transforming enzymes. Furthermore, any insert that lacks an
the recombinant pETBlue plasmid into the host internal stop codon can be cloned in-frame with
strains Tuner™(DE3)pLacI followed by induc- the C-terminal HSV•Tag® epitope and His•Tag®
tion with IPTG. Other (DE3)pLacI host strains sequences.
8
EcoR V EcoR V
Vector Insert Vector Insert

T7 promoter RBS Met T7 promoter RBS Met
AAGGAGATATAGAT ATG XXX AAGGAGATATAGAT G XXX
9 TTCCTCTATATCTA TAC XXX
8 bp spacing between RBS and ATG

TTCCTCTATATCTA C XXX
6 bp spacing between RBS and ATG
pETBlue-1: Two alternative designs of insert 5'-ends for optimal expression
10
3-ORF site 3-ORF site
Nco I EcoR V Sma I Ecl136 II Pvu II Bst1107 I Pml I
CCATGGCGATATCCCGGGAGCTC...MCS...GCACAGCTGTATACACGTGCA...HSV•Tag/His•Tag
MetAlaIleSerArgGluLeu... ...AlaGlnLeuTyrThrArgAla...
11 pETBlue-2: Convenient restriction sites for reading frame adjustment at both ends of pETBlue-2 inserts
Shown are the 5' and 3' “3-ORF” regions of the multiple cloning sites (MCS) in pETBlue-2. The amino acid sequence of T7-driven expressed
proteins is shown, including the reading frame for C-terminal HSV•Tag/His•Tag fusions.
Appendices
Indices

Novagen
Protein Expression
pETBlue™ PCR Cloning Kits pETBlue™ AccepTor™ Vector Kits

1
Convenient cloning of PCR-amplified DNA directly into pETBlue Vectors Product Size Cat. No.
Introductory pETBlue-1
In addition to the pETBlue™ Systems, which such as those amplified with non-proofreading AccepTor Vector Kit 10 rxn 70597-3
contain uncut plasmids, Novagen offers the DNA polymerases (e.g., NovaTaq™ DNA pETBlue-1 AccepTor 20 rxn 70598-3
pETBlue vectors in formats ready for insertion Polymerase), while the Perfectly Blunt® Cloning Vector Kit 40 rxn 70598-4
of PCR products. Two types of kits are available;
the AccepTor™ Vector Kits enable direct inser-
Kits are designed for cloning inserts having any
type of end.
pETBlue-1 AccepTor
Vector (linearized vector)
20 rxn
40 rxn
70599-3
70599-4
2
tion of DNA containing single 3'-dA overhangs,
pETBlue Perfectly Blunt® Cloning Kits
Product Size Cat. No.
pETBlue-1 AccepTor Vector
Perfectly Blunt 3
EcoR I
Sma I
Srf I
dU
dU Primer design for expression of inserts in Cloning Kit 10 rxn 70633-3
pETBlue-1 AccepTor Vector
pETBlue-1 Perfectly 20 rxn 70634-3
la cZ tet Blunt Cloning Kit 40 rxn 70634-4
Amplification with sense primers beginning with ATG at their 5'-end
lac rrnB terminator
T7 will ensure optimal spacing between the RBS and translation initia- pETBlue-1 Blunt 20 rxn 70620-3
T7 terminator
tion sites for efficient protein synthesis in E. coli. There are no
restrictions on the design of primers that specify the carboxyl termi-
Vector
(linearized vector)
40 rxn
500 rxn
70620-4
70620-5 4
nus of the target sequence.
Met...
ori
pETBlue-1 Perfectly Blunt

3,476 bp Sense primer: 5'-ATGXXX...
Cloning Kit 10 rxn 70635-3
Antisense primer: No restrictions
pETBlue-2 Perfectly 20 rxn 70636-3
gi
Blunt Cloning Kit 40 rxn 70636-4
5
n
Ap i
or pETBlue-2 Blunt 20 rxn 70621-3
f1
Vector 40 rxn 70621-4
(linearized vector) 500 rxn 70621-5
Components:
Please see Chapter 2, Cloning for components
pETBlue-1 and pETBlue-2 Blunt Vectors

and additional information. 6
pETBlue-1 Additional Information Available
EcoR I
Sma I
Srf I
AccepTor Vector Kits Protocol TB248

Perfectly Blunt Cloning Kits Manual TB183
pET System Manual TB055
7
Sse8387 I
Bst1107 I
pETBlue-2
Ecl136 II
BamH I
pETBlue System Protocol TB249

BssH II
Hind III
EcoR I
BsrG I
Nsp V
Sma I
Pvu II
Nco I
Kpn I
Aat II
Xho I
Pml I
Eag I
Pac I
Asc I
Mlu I
Not I
Cla I
Pst I
Sal I
HSV•Tag His•Tag Vector Cloning Region Sequences Appendix

Patent and Licensing Information p. 278
la cZ tet
lac rrnB terminator
T7
T7 terminator
8
pETBlue-1
ori
pETBlue-2
i
9
n
Ap ig
or
f1
Primer design for expression of inserts in pETBlue-1 and pETBlue-2 Blunt Vectors
pETBlue-1: Amplification with sense primers beginning with ATG at should begin with the third base of the Ile/Met codon. To express a
10
their 5'-end will ensure optimal spacing between the RBS and trans- target protein fused with C-terminal HSV•Tag® and His•Tag® pep-
lation initiation sites for efficient protein synthesis in tides, the antisense primer should begin with two bases in any combi-
E. coli. There are no restrictions on the design of primers that specify nation except TA or CA, and specify an antisense codon beginning with
the carboxyl terminus of the target sequence. the third base.
Sense primer:
Met...
5'-ATGXXX...
MetAlaIle....insert.....Ser
Vector: ATGGCGATNXXX...........ATCC 11
Antisense primer: No restrictions TACCGCTA..........YYYNNTAGG
pETBlue-2 encodes an ATG start codon 8 base pairs downstream of Sense primer: 5'-NXXX......
the T7 gene 10 RBS. The blunt cloning site is located such that the if N = G, Met codon is generated instead of Ile.
Appendices
insert will specify the fourth amino acid following Met-Ala-Ile at the N- Antisense primer: 5'-NNYYY.....
terminus of the expressed protein. In order to achieve the correct if NN = CA or TA, a stop codon is generated in sense strand.
Indices
reading frame, the 5'-end of the insert (and the sense PCR primer)

87
Novagen
Protein Expression
1 pET System Tutorial

The premier E. coli expression system
The pET System is the most powerful system Figure 1. Control elements of the pET System
yet developed for the cloning and expression of
recombinant proteins in E. coli. Based on the T7 IPTG Induction IPTG Induction
2 promoter-driven system originally developed by
Studier and colleagues (1–3), Novagen’s pET
E. coli RNA
polymerase
T7 RNA
polymerase
T7 gene 1 Target gene

System has been used to express thousands of T7 RNA polymerase
different proteins. Please refer to pET System
Overview earlier in this chapter for the basic lac o lac o
lac promoter T7 promoter
features of the pET System. This section
3 describes some of the unique characteristics of
DE3
INACTIVE
the system in greater detail.

lac lac
Control Over Basal Expression Levels repressor repressor
pET
The pET System provides six possible
lac I gene lac I gene
4 vector-host combinations that enable tuning of
basal expression levels to optimize target gene
T7 lysosyme
pLysS
expression (2). These options are necessary or E
T7 lysozyme
because no single strategy or condition is suit- gene
able for every target protein.
5
Host Strains
After plasmids are established in a non-
E. coli genome HOST CELL
expression host, they are most often trans-
formed into a host bearing the T7 RNA poly- Table 1. pET System Host Strains
merase gene (lDE3 lysogen) for expression of
Available as
target proteins. Figure 1 illustrates in schematic
Competent
6 form the host and vector elements available for
control of T7 RNA polymerase levels and the
Strain Derivation Key Feature(s) Antibiotic Resistance Cells
AD494 K-12 trxB mutant; facilitates cytoplasmic Kan yes
subsequent transcription of a target gene in a AD494(DE3) disulfide bond formation Kan yes
pET vector. In lDE3 lysogens, the T7 RNA poly- AD494(DE3)pLysS Kan + Cam yes
merase gene is under the control of the lacUV5 BL21 B834 Lacks lon and ompT none yes
promoter. This allows some degree of tran- BL21(DE3) proteases none yes
7 scription in the uninduced state and in the

BL21(DE3)pLysS
BL21(DE3)pLysE
Cam
Cam
yes
no
absence of further controls is suitable for BL21trxB(DE3) BL21 BL21 trxB mutant; facilitates Kan yes
expression of many genes whose products have BL21trxB(DE3)pLysS cytoplasmic disulfide bond formation Kan + Cam yes
innocuous effects on host cell growth. For more BLR BL21 BL21 recA mutant; stabilizes tandem Tet yes
stringent control, hosts carrying either pLysS or BLR(DE3) repeats Tet yes
BLR(DE3)pLysS Tet + Cam yes
pLysE are available. The pLys plasmids encode
8 T7 lysozyme, which is a natural inhibitor of T7 B834
B834(DE3)
B strain met auxotroph; 35S-met and
selenomethionine labeling
none
none
no
yes
RNA polymerase, and thus reduces its ability to B834(DE3)pLysS Cam yes
transcribe target genes in uninduced cells. pLysS HMS174 K-12 recA mutant Rif yes
hosts produce low amounts of T7 lysozyme, HMS174(DE3) Rif resistance Rif yes
while pLysE hosts produce much more enzyme HMS174(DE3)pLysS Rif + Cam yes
9 and, therefore, represent the most stringent con-

trol available in lDE3 lysogens (4).
HMS174(DE3)pLysE
NovaBlue K-12 recA, endA, lacIq
Rif + Cam
Tet
no
yes
NovaBlue(DE3) cloning, plasmid preps Tet yes
Thirteen different host strains
Origami™ K-12 trxB/gor mutant, greatly facilitates Kan + Tet yes
Thirteen different host strains are available Origami(DE3) cytoplasmic disulfide bond formation Kan + Tet yes
as lDE3 lysogens (see Table 1). The most widely Origami B(DE3)pLysS Kan + Tet + Cam yes
10 used hosts are BL21 and its derivatives, which

have the advantage of being deficient in both lon
Origami B
Origami(DE3)
Tuner BL21 lacY deletion, trxB/gor mutant,
greatly facilitates cytoplasmic disulfide
Kan + Tet
Kan + Tet
yes
yes
Origami B(DE3)pLysS bond formation; allows precise control Kan + Tet + Cam yes
(5) and ompT proteases. The B834 strain is a with IPTG Kan + Tet + Cam yes
methionine auxotroph and, therefore, enables Rosetta™ Tuner Enhances expression of Cam yes
high specific activity labeling of target proteins Rosetta(DE3) proteins having codons rarely used in Cam yes
with 35S-methionine or selenomethionine (6). Rosetta(DE3)pLysS E. coli, lacY deletion mutant Cam yes
11 The BLR strain is a recA– derivative that
improves plasmid monomer yields and may help
Rosetta-gami™
Rosetta-gami(DE3)
Origami Enhances expression of
proteins having codons rarely used in
Kan + Tet + Cam
Kan + Tet + Cam
yes
yes
Rosetta-gami(DE3)pLysS E. coli, trxB/gor mutant Kan + Tet + Cam yes
stabilize target plasmids containing repetitive
RosettaBlue™ NovaBlue Enhances expression of Tet + Cam yes
sequences. Two thioredoxin reductase (trxB) RosettaBlue(DE3) proteins having codons rarely used in Tet + Cam yes
Appendices
mutant strains (AD494, BL21trxB) are available RosettaBlue(DE3)pLysS E. coli, recA, endA, lacIq Tet + Cam yes
Indices
that facilitate disulfide bond formation in the E. Tuner™ BL21 BL21 lacY deletion mutant none yes
coli cytoplasm (7). The Origami™, Origami B Tuner(DE3) allows precise control with IPTG none yes
continued on next page Tuner(DE3)pLysS Cam yes

Novagen
Protein Expression
pET System Tutorial continued pET System Advantages

1
Powerful
Recombinant antibody Human p53 • Highest expression levels, tightest control
Promoter T7lac T7 T7lac T7lac T7lac T7 T7 T7lac T7lac over basal expression
Host BL21(DE3) – pLysE – pLysS pLysE – pLysS – pLysS • Choice of vectors and host strains to
(uninduced)
kDa control basal and induced expression levels 2
–150 • Precise control of induced expression with
IPTG in Tuner™ and Rosetta™ hosts
–100
–75 • Rare codons supplied by Rosetta hosts
p53 → • Origami™ strains for enhanced disulfide
–50
–35
bond formation in the cytoplasm 3
rec. ab →
–25 Versatile
• Choice of N-terminal and C-terminal fusion
tags for detection, purification and
–15
localization
• Expanded multiple cloning sites in all three
4
reading frames
Figure 2. Effect of vector/host combination on expression levels of two proteins • f1 origin of replication for mutagenesis and
The indicated cell cultures were grown at 37°C to OD600 of approximately 0.8 and expression induced with 1 mM IPTG for 2.5 h. Total sequencing
cell protein samples were run along with Novagen’s Perfect Protein™ Markers on a 4–20% SDS polyacrylamide gradient gel followed by
staining with Coomassie blue. Vectors used were pET-20b(+) and pET-22b(+) for the recombinant antibody and pET-23b(+) and
pET-21b(+) for p53.
Rapid
• E. coli-based system for rapid results
5
• Convenient restriction sites for subcloning
from other vectors
and Rosetta-gami™ strains are double mutants diately downstream from the 17 bp promoter
• Choice of methods for one-step purification
of trxB/gor, which are the key enzymes in both region. Binding of the lac repressor at this site
major reductive pathways (8). These hosts thus effectively reduces transcription by T7 RNA
of target proteins without antibodies
6
represent a significant advantage for the forma- polymerase, thus providing a second lacI-based Complete
tion of properly folded disulfide-containing pro- mechanism (besides the repression at lacUV5) to
• Variety of system configurations plus many
teins. The Rosetta™, Rosetta-gami, and suppress basal expression in lDE3 lysogens.
supporting products
RosettaBlue™ strains supply the tRNAs for six pET plasmids with the T7lac promoter also carry
codons used only rarely in E. coli, which allevi-
ates poor expression caused by incompatible
their own copy of lacI to ensure that enough
repressor is made to titrate all available operator 7
codon usage in some eukaryotic genes (9). The sites.
K-12 derivative strains HMS174, NovaBlue, and In practice, it is usually worthwhile to test
RosettaBlue are recA–, like BLR. These strains several different vector/host combinations to
may stabilize certain target genes whose prod- obtain the best possible yield of protein in its
ucts may cause the loss of the DE3 prophage.
NovaBlue and RosettaBlue are potentially useful
desired form (10). Figure 2 illustrates dramatic
differences in the expression of two target pro-
8
as stringent hosts due to the presence of the high teins with various combinations.
affinity lacIq repressor encoded by the F epi-
Control Over Induced Expression Levels
some. In addition, Novagen offers the lDE3
Lysogenization Kit for making new expression In many cases the expression of optimal
hosts with other genetic backgrounds. An alterna- levels of active, soluble protein depends on 9
tive for expressing extremely toxic genes or host cell background, culture conditions, and
preparing a new lDE3 lysogen is to provide T7 vector configuration; often the conditions for
RNA polymerase by infection with lCE6. highest activity of a target protein do not cor-
Although not as convenient as inducing a lDE3 relate with conditions that produce the high-
lysogen with IPTG, this strategy may be preferred
for certain applications.
est mass of target protein. In addition to
offering variable stringency based on
10
High Stringency T7lac Promoter vector/host combinations that provide control
over basal expression of T7 RNA polymerase,
In addition to the choice of three basic
the pET System offers precise control over
expression stringencies at the host level, the
target protein expression based on inducer
pET system provides two different stringency
options at the level of the T7 promoter itself: the
(IPTG) concentration, made possible by the
lacY mutation in the Tuner™, Rosetta, and
11
“plain” T7 promoter and the T7lac promoter (8;
Origami B host strains.
also shown in Figure 1). The T7lac promoter
contains a 25 bp lac operator sequence imme-
Appendices
Indices

89
Novagen
Protein Expression
1 pET System Tutorial continued
Choosing a pET Vector lowing purification by digestion with a site-spe-

cific protease. The LIC (ligation-independent T7
A wide variety of pET vectors is available. All
cloning) strategy is especially useful for this p
A
are derived from pBR322 and vary in leader
2 sequences, expression signals, fusion tags, rele-
approach, because the cloning procedure
enables the removal of all amino terminal vector-
vant restriction sites, and other features. There pET-3a–d
encoded sequences with either enterokinase or
are two major categories of pET plasmids pET-12a–c
Factor Xa .
known as transcription vectors and translation pET-14b
Cloning strategies can affect the choice of pET-17b
vectors:
vector due to the need for restriction site and pET-17xb
3 • The transcription vectors [including pET-
21(+), pET-23(+), and pET-24(+)] express
reading frame compatibilities. Because many of
the pET vectors share common restriction site
target RNA but do not provide translation or
configurations, it is usually possible to clone a i
signals. They are useful for expressing
target gene into several vectors with a single
proteins from target genes that carry their
preparation of the insert. Different considera-
own bacterial translation signals. Note that
4 the transcription vectors can be identified
by lack of a letter suffix after the name.
tions apply when using PCR cloning strategies.
The LIC Vector Kits are recommended for this
purpose, and enable the preparation of inserts
• The translation vectors contain efficient
by PCR and eliminate the need for restriction
translation initiation signals and are designed
digestion of vector or insert. The Appendix con-
for protein expression. Most contain cloning
tains sequences of cloning and expression T7
an
5 sites in reading frames a, b, or c that
K
regions for the pET vectors.
correspond to the GGA, GAT, or ATC triplet
of the BamH I site, respectively. Solubility and Cellular Localization
Once you have considered your application
Primary Considerations
and cloning strategy, a good starting point for
Choosing a pET vector for expression usual- pET-9a–d
any expression project is to determine the cellu-
6 ly involves a combination of factors. Consider
the following three primary factors:
lar localization and solubility of the target pro-
tein. In many applications, it is desirable to
• The application intended for the expressed express proteins in their soluble, active form.
protein Solubility of a particular target protein is ri
• Specific information known about the determined by a variety of factors, including the o
expressed protein individual protein sequence. In most cases, solu-
7 • Cloning strategy bility is not an all-or-none phenomenon; the vec-
tor, host, and culture conditions can be used to
Applications for proteins expressed in pET
increase or decrease the proportion of soluble
vectors vary widely. For example, analytical
and insoluble forms obtained. The choice of vec-
amounts of a target protein may be needed for f1 origin
tor and expression host can significantly
activity studies, screening and characterizing
increase the activity and amount of target pro- T
8 mutants, screening for ligand interactions, and
antigen preparation. Large amounts of active
tein present in the soluble fraction. A vector can
A
p
7
enhance solubility and/or folding in three ways:
protein may be required for structural studies,
1, provide for fusion to a polypeptide that itself
use as a reagent, or affinity matrix preparation.
is highly soluble (e.g., GST, thioredoxin, NusA), pET-20b(+)
Any number of vectors may be suitable for
2, provide for fusion to an enzyme that catalyzes pET-23(+)
expression of analytical amounts of protein for
9 screening or antigen preparation, yet only one
disulfide bond formation (e.g., thioredoxin,
DsbA, DsbC), or 3, provide a signal sequence for
pET-23a–d(+)
combination of vector, host strain, and culture

translocation into the periplasmic space. When
conditions may work best for large scale purifi-
using vectors designed for cytoplasmic expres- ri
cation. If a high yield of active protein is needed
o
sion, folding can be improved in hosts that are

on a continual basis, it is worth testing a matrix
permissive for the formation of disulfide bonds
10 of vector, host, and culture combinations to find
the optimal result.
in the cytoplasm. The thioredoxin reductase
(trxB) mutation has been shown to allow the
Any information available about the target pET vector backbones: “plain” T7 promoter vectors
formation of disulfide bonds in the E. coli cyto-
protein may help determine the choice of vector. The pET vectors shown here and on the facing page are grouped
plasm, which is further enhanced by the addi-
For example, some proteins require no extrane- according to the functional elements present on the plasmid back-
tional mutation in the glutathione reductase bones. Features of the T7 cloning and expression regions (indicated
ous sequence on one or both termini for activity.
11 Most pET vectors enable cloning of unfused
sequences; however, expression levels may be
(gor) gene in Origami™ and Rosetta-gami™
hosts (8). Induction at lower temperatures
here as “T7” and “T7lac”) are shown on the following pages and in the
Appendix. Note that the pET vector sequences are numbered by the
(15°–25°C) can also increase the proportion of pBR322 convention, so that the T7 expression region is reversed on
affected if a particular translation initiation these maps and in the published sequences.
soluble target proteins.
sequence is not efficiently utilized in E. coli. In
The pET-43.1 and pET-32 vectors incorporate
Appendices
these cases, an alternative is to construct a

fusion tags specifically designed to enhance the
Indices
fusion protein with efficiently expressed amino

solubility of target proteins in the E. coli
terminal sequences (available with many pET
vectors) and then remove the fusion partner fol-

Novagen
Protein Expression
pET System Tutorial continued

1
cytoplasm. These vectors are also compatible Fusion Tags for Different Needs
with trxB mutant hosts AD494 and BL21trxB, T7
If a fusion sequence is tolerated by the appli- la
p
and with the trxB/gor mutant Origami™,
c
cation you are using, it is useful to produce
Origami B, and Rosetta-gami™ strains. The pET-
43.1a–c(+) vectors incorporate the 495 aa solu-
fusion proteins carrying the S•Tag™, T7•Tag®,
GST•Tag, His•Tag®, or HSV•Tag® peptides for
2
bility-promoting NusA (Nus•Tag™) sequence, easy detection on Western blots. These peptides pET-11a–d
which was discovered through a systematic are small in size (except for the GST•Tag pET-15b
search for E. coli proteins that have the highest pET-16b
sequence) and the detection reagents for them
la cI
potential for solubility when overexpressed (11). pET-19b
are extremely specific and sensitive. The
Many proteins that are normally produced in an His•Tag, GST•Tag, S•Tag, and T7•Tag 3
insoluble form in E. coli tend to become more sequences can also be used for affinity purifica-
ri
o
soluble when fused with the 109 aa N-terminal tion using the corresponding resins and buffer
thioredoxin (Trx•Tag™) sequence. The Trx•Tag kits.
expressed from pET-32 vectors not only Fusion proteins can be accurately quantified
enhances the solubility of many target proteins,
but appears to catalyze the formation of disul-
in crude extracts or purified form using S•Tag
and GST•Tag Assay Kits. The FRETWorks™
4
fide bonds in the cytoplasm of trxB mutants S•Tag Assay Kit is based on a novel substate
(12). that enables fluorescent detection of less than
Schistosomal glutathione-S-transferase (GST) 1 fmol of fusion protein in a homogeneous for-
is commonly used as an N-terminal fusion part- f1 origin
mat. (See Chapter 7, Protein & Gene Analysis.)
ner when expressing proteins in E. coli. The His•Tag sequence is very useful as a p
T7
la 5
c
Although not specifically designed for this pur- fusion partner for purification of proteins in gen-
pET-21(+)
pose, the 220 aa GST•Tag™ sequence has been eral. It is especially useful for those proteins ini- pET-21a–d(+)
reported to enhance the solubility of its fusion tially expressed as inclusion bodies, because pET-22b(+)
partners. The pET-41 and -42 series of vectors affinity purification can be accomplished under pET-25b(+)
encode the GST•Tag sequence driven by the totally denaturing conditions that solubilize the
powerful T7lac promoter. Note that these vec-
pET-31b(+)
pET-32a–c(+) 6
lac I
protein.
tors carry kanamycin resistance so are not rec- The CBD•Tag™ sequences are also generally pET-32 Ek/LIC
ommended for use with trxB mutant hosts. pET-32 Xa/LIC
useful for low cost affinity purification. They are
An alternative strategy to obtain active, solu- pET-43.1a–c(+)*
also uniquely suited to refolding protocols [espe- o ri
ble proteins is to use vectors that enable export cially pET-34b(+) and 35b(+), which contain the
into the periplasm, which is a more favorable
environment for folding and disulfide bond for-
CBDclos•Tag sequence]. Because only properly
refolded CBDs bind to the cellulose matrix, the
* Ap gene in opposite orientation in
pET-43.1 series
7
mation. For this purpose vectors carrying signal CBIND™ affinity purification step can remove
peptides are used. DsbA and DsbC are periplas- improperly folded molecules from the prepara-
mic enzymes that catalyze the formation and iso- tion. While any of the tags can be used to immo-
merization of disulfide bonds, respectively. The bilize target proteins, the CBD•Tag sequences f1 origin
208 aa DsbA•Tag™ [pET-39b(+)] and 236 aa
DsbC•Tag™ [pET-40b(+)] vectors enable fusion
are ideally suited for this purpose due to the
an
T7
l a 8
inherent low non-specific binding and biocom- c
K
pET-24(+) pET-34b(+)
of target polypeptides to these enzymes, which patibility of the cellulose matrix. pET-24a–d(+) pET-35b(+)
include their N-terminal secretion signals. If the The Nus•Tag, Trx•Tag and GST•Tag pET-26b(+) pET-36b(+)
fusion protein is exported to the periplasm, the sequences have been reported to enhance the pET-27b(+) pET-37b(+)
Dsb partner can assist in proper disulfide bond pET-28a–c(+) pET-38b(+)
formation. Other pET vectors that carry signal
solubility of their fusion partners. The Nus•Tag
and Trx•Tag vectors are compatible with pET-29a–c(+) pET-39b(+) 9
l acI
sequences without the additional DsbA or DsbC pET-30a–c(+) pET-40b(+)

Origami, Origami B, and Rosetta-gami host
coding regions are also available. pET-30 LIC pET-41a–c(+)
strains, which facilitate disulfide bond formation
pET-33b(+) pET-42a–c(+)
Some purification strategies optimize pro- in the cytoplasm.
ri
o
duction of insoluble inclusion bodies in the cyto- The various fusion tags available and their
plasm. Inclusion bodies are extracted and solubi-
lized; then the target protein is refolded in vitro,
corresponding pET vectors are listed in the fol-
lowing table. A number of pET vectors encode
10
(e.g., with Novagen’s Protein Refolding Kit). This several of the fusion tags in tandem as amino-
procedure usually produces the highest yields of terminal fusion partners. In addition, many vec-
initial protein mass and protects against prote- tors enable expression of fusion proteins carry- pET vector backbones:
olytic degradation in the host cell. However, the ing a peptide tag on each end by allowing in- T7lac promoter vectors
efficiency of refolding into active protein varies
significantly with the individual protein and can
frame read-through of the target gene sequence. 11
Using vectors with protease cleavage sites
be quite low. The pET-31b(+) vector is specifi- (thrombin, Factor Xa, enterokinase) between the
cally designed for the generation of insoluble amino terminal tag and the target sequence
Appendices
fusion proteins and provides a powerful method enables optional removal of one or more tags
Indices
for the production of small proteins and pep- following purification. Vectors that represent a
tides. continued on next page

91
Novagen
Protein Expression
1 pET System Tutorial continued
good selection for cellular localization and affin- Table 2. Fusion tags available for pET constructs
ity tag configurations are pET-30 Ek/LIC, pET-32
Ek/LIC, pET-41 Ek/LIC and pET-43.1 Ek/LIC. A N/C
2 single preparation of insert can be used with all

of the ready-to-use Ek/LIC vectors to allow con- Tag
Terminal
or Internal Size (aa)
Basis for Detection
and/or Purification Applications pET Vector Series
venient construction of several target gene con- T7•Tag® N, I 11 or 260 monoclonal antibody Western blot, 3, 9, 11, 17 17x,
figurations at once. immunoprecipitation, 21, 23, 24, 28, 33
purification
How to Order S•Tag™ N, I 15 S-protein (104 aa) affinity Western blot, 29, 30, 32, 34–37
3 The pET Vectors are available configured quantitative assay,
purification
39, 40, 41, 42,
43.1
into Expression Systems containing one or more
vectors, plus glycerol stocks and competent His•Tag® N, C, I 6, 8, or 10 metal chelation His•Bind® resin 14-16, 19–43.1
chromatography purification
cells of popular host strains for cloning and
(native or denaturing)
expression. Individual plasmids and cell strains
HSV•Tag® C 11 monoclonal antibody Western blot, 25, 27, 43.1
4 are available separately.
1. Moffatt, B. A., and Studier, F. W. (1986) J. Mol.
immunofluorescence
Biol. 189, 113–130.

pelB/ompT N 20/22 potential periplasmic protein export/folding 12, 20, 22
2. Rosenberg, A. H., Lade, B. N., Chui, D., Lin, S., localization 25, 26, 27
Dunn, J. J., and Studier, F. W. (1987) Gene 56,
125–135.
5 3. Studier, F. W., Rosenberg, A. H., Dunn, J. J., and

Dubendorff, J. W. (1990) Methods Enzymol. 185,
60–89.
KSI N 125 highly expressed
hydrophobic domain
small protein/peptide
production/purification,
insoluble protein
31
4. Studier, F. W. (1991) J. Mol. Biol. 219, 37–44. Trx•Tag™ N 109 thioredoxin soluble protein, cyto- 32
5. Phillips, T. A., Van Bogelen, R. A., and Neidhardt, F. plasmic disulfide bond
C. (1984) J. Bacteriol. 159, 283–287. formation in trxB– hosts
6 6.
7.
Leahy et al. (1992) Science 258, 987–991.
Derman, A. I., Prinz, W. A., Belin, D., and Beckwith,
PKA site N 5 protein kinase A
recognition site
in vitro phosphorylation 33
J. (1993) Science 262, 1744–1747.

8. Dubendorff, J. W. and Studier, F. W. (1991) J. Mol. CBDclos•Tag N 156 polyclonal antibody, Western blot, 34, 35
Biol. 219, 45–59. cellulose binding purification, noncovalent
9. Novy, R., Drott, D. Yaeger, K., and Mierendorf, R. domain immobilization
7 (2001) inNovations 12, 1-3.

10. Mierendorf, R., Yaeger, K., and Novy, R. (1994)
CBDcenA•Tag N 114 polyclonal antibody,
cellulose binding domain,
protein export, Western
blot, purification, non-
36, 37
inNovations 1, 1–3. periplasm/media covalent immobilization

11. Harrison, R. (2000) inNovations 11, 4–7 CBDcex•Tag C 107 polyclonal antibody, protein export, Western 38
12. Stewart et al. (1998) EMBO J. 17, 5543–5550. cellulose binding domain, blot, purification, non-
periplasm/media covalent immobilization
8 Dsb•Tag™ N 208 (DsbA) potential periplasmic

236 (DsbC) localization
soluble protein, peri-
plasmic disulfide bond
39, 40
formation, isomerization
GST•Tag™ N 220 glutathione affinity purification, 41, 42
monoclonal antibody Western blot,
enzymatic activity quantitative assay
9 Nus•Tag™ N 495 NusA soluble protein, cyto- 43.1
plasmic disulfide bond
formation in trxB– hosts
10
11
Appendices
Indices

Novagen
Protein Expression
pET Vector DNA 1

Individual plasmid DNAs
The pET Vectors are supplied as 10 µg puri- Vector Cat. No. Vector Cat. No.
fied plasmid DNA. Each order also includes an
pET-3a DNA 69418-3 pET-24d(+) DNA 69752-3
Induction Control strain (supplied as a glycerol
stock). Complete sequences and detailed vector
maps are available on the Novagen web site:
pET-3b DNA
pET-3c DNA
69419-3
69420-3
pET-25b(+) DNA
pET-26b(+) DNA
69753-3
69862-3
2
www.novagen.com
pET-3d DNA 69421-3 pET-27b(+) DNA 69863-3
pET-9a DNA 69431-3 pET-28a(+) DNA 69864-3
pET-9b DNA 69432-3 pET-28b(+) DNA 69865-3
pET-9c DNA 69433-3 pET-28c(+) DNA 69866-3 3
pET-9d DNA 69434-3 pET-29a(+) DNA 69871-3
pET-11a DNA 69436-3 pET-29b(+) DNA 69872-3
pET-11b DNA 69437-3 pET-29c(+) DNA 69873-3
pET-11c DNA 69438-3 pET-30a(+) DNA 69909-3 4
pET-11d DNA 69439-3 pET-30b(+) DNA 69910-3
pET-12a DNA 69440-3 pET-30c(+) DNA 69911-3
pET-12b DNA 69441-3 pET-31b(+) DNA 69952-3
pET-12c DNA
pET-14b DNA
69442-3
69660-3
pET-32a(+) DNA
pET-32b(+) DNA
69015-3
69016-3
5
pET-15b DNA 69661-3 pET-32c(+) DNA 69017-3
pET-16b DNA 69662-3 pET-33b(+) DNA 69054-3
pET-17b DNA 69663-3 pET-34b(+) DNA 70102-3
pET-17xb DNA 69664-3 pET-35b(+) DNA 70103-3
6
pET-19b DNA 69677-3 pET-36b(+) DNA 70133-3
pET-20b(+) DNA 69739-3 pET-37b(+) DNA 70135-3
pET-21(+) DNA 69770-3 pET-38b(+) DNA 70137-3
pET-21a(+) DNA 69740-3 pET-39b(+) DNA 70090-3 7
pET-21b(+) DNA 69741-3 pET-40b(+) DNA 70091-3
pET-21c(+) DNA 69742-3 pET-41a(+) DNA 70556-3
pET-21d(+) DNA 69743-3 pET-41b(+) DNA 70557-3
pET-22b(+) DNA 69744-3 pET-41c(+) DNA 70558-3 8
pET-23(+) DNA 69771-3 pET-42a(+) DNA 70561-3
pET-23a(+) DNA 69745-3 pET-42b(+) DNA 70562-3
pET-23b(+) DNA 69746-3 pET-42c(+) DNA 70563-3
pET-23c(+) DNA 69747-3 pET-43.1a(+) DNA 70939-3
pET-23d(+) DNA 69748-3 pET-43.1b(+) DNA 70940-3
9
pET-24(+) DNA 69772-3 pET-43.1c(+) DNA 70941-3
pET-24a(+) DNA 69749-3 pLysE DNA (1µg) 69658-3
pET-24b(+) DNA 69750-3 pLysS DNA (1µg) 69659-3
pET-24c(+) DNA 69751-3 pLacI DNA (1µg) 70610-3 10
Additional Information Available
inNovations Nos. 1, 3, 7–9, 11
Vector Cloning Region Sequences
Vector maps and sequences
Appendix
www.novagen.com
11
Appendices
Indices

93
Novagen
Protein Expression
1 pET Expression Systems

pET vectors plus host strains, with Induction Control
pET Expression Systems provide core System Vector(s) Fusion Tag(s) Cat. No.
reagents needed for target gene cloning and
pET Expression System 3 pET-3a–d T7•Tag® 69409-3
expression. System 3 plus Competent Cells 69410-3
2 Components:
• pET vector DNA, 10 µg each of the indicated
pET Expression System 9 pET-9a–d T7•Tag 69415-3
System 9 plus Competent Cells 69416-3
plasmids
pET Expression System 11 pET-11a–d T7•Tag 69405-3
• Host bacterial strains BL21, BL21(DE3), and System 11 plus Competent Cells 69406-3
BL21(DE3)pLysS, glycerol stocks
pET Expression System 12 pET-12a–c 69407-3
3 •
•
Induction control clone, glycerol stock
All vector sequences and maps are available
System 12 plus Competent Cells
pET Expression System 14b pET-14b His•Tag®
69408-3
70753-3
at www.novagen.com System 14b plus Competent Cells 70754-3
Systems Plus Competent Cells contain a pET Expression System 15b pET-15b His•Tag 70755-3
set of three host strains ready for high-efficiency
System 15b plus Competent Cells 70756-3
4 transformation of pET recombinants. These

include the following additional components,
pET Expression System 16b
System 16b plus Competent Cells
pET-16b His•Tag 70757-3
70758-3
which are sufficient for up to 10 transformations pET Expression System 17b pET-17b T7•Tag 69726-3
in each host: System 17b plus Competent Cells 69731-3
Components: pET Expression System 17xb pET-17xb T7•Tag 69727-3
• One 0.2 ml aliquot of each host NovaBlue, System 17xb plus Competent Cells 69732-3
5 BL21(DE3), and BL21(DE3)pLysS as
pretested competent cells
pET-19b His•Tag 70759-3
70760-3
• SOC Medium pET Expression System 20b pET-20b(+) His•Tag 70761-3
• Test Plasmid System 20b plus Competent Cells 70762-3
pET Expression System 21 pET-21a–d(+) T7•Tag, His•Tag 70763-3
Purification and Detection Reagents are
6 available separately. A list of affinity purification
pET Expression System 22b pET-22b(+) His•Tag
70764-3
70765-3
resins and buffer kits is on the following page.
Please refer to Chapters 7, Protein & Gene
Analysis and 9, Protein Purification for complete pET Expression System 23 pET-23a–d(+) T7•Tag, His•Tag 70767-3
descriptions and ordering information.
pET Expression System 24 pET-24a–d(+) T7•Tag, His•Tag 70769-3
7 pET CBD Fusion Systems provide core System 24 plus Competent Cells 70770-3
reagents needed for target gene cloning, expres- pET Expression System 25b pET-25b(+) His•Tag, HSV•Tag® 70771-3
sion, and CBIND™ purification. System 25b plus Competent Cells 70772-3
Components: pET Expression System 26b pET-26b(+) His•Tag 70773-3
• pET vector DNA, 10 µg System 26b plus Competent Cells 70774-3
8 • Host bacterial strains BL21, BL21(DE3), and
pET-27b(+) His•Tag, HSV•Tag® 70775-3
70776-3
• Induction control clone, glycerol stock product listing continued on next page
• CBIND 300 Cartridges, 5 each
• CBIND 900 Cartridges, 5 each Additional Information Available
9 • CBIND Elute Reagent, 10 ml pET System Manual
inNovations
TB055
Nos. 1, 3, 7–9, 11–13
• All vector sequences and maps are available
Vector Cloning Region Sequences Appendix
at www.novagen.com Vector maps and sequences www.novagen.com
10
11
Appendices
Indices

Novagen
Protein Expression
pET Expression Systems continued

1
System Vector(s) Fusion Tag(s) Cat. No.
pET Expression System 28 pET-28a–c(+) His•Tag®, T7•Tag® 70777-3
pET Expression System 29 pET-29a–c(+) His•Tag, S•Tag™ 70779-3 2
pET Expression System 30 pET-30a–c(+) His•Tag, S•Tag 70781-3
pET Expression System 31b pET-31b(+) His•Tag 70783-3
includes BLR series host strains in place of the BL21 series hosts
70784-3
3
pET Trx Fusion System 32 pET-32a–c(+) His•Tag, S•Tag 70785-3
includes AD494 series host strains in addition to the BL21 series hosts
pET Expression System 33b pET-33b(+) His•Tag, T7•Tag 70787-3

System 33b plus Competent Cells 70788-3 4
pET CBD Fusion System 34b pET-34b(+) His•Tag, S•Tag, 70110-3
System 34b plus Competent Cells CBD•Tag™ 70112-3
System 35b plus Competent Cells CBD•Tag 70113-3
pET CBD Fusion System 36b
pET-36b(+) His•Tag, S•Tag,
CBD•Tag
70146-3
70148-3 5
System 37b plus Competent Cells CBD•Tag 70150-3
pET CBD Fusion System 38b pET-38b(+) His•Tag, CBD•Tag 70151-3
pET Dsb Fusion System 39b pET-39b(+) His•Tag, S•Tag 70789-3 6
pET Dsb Fusion System 40b pET-40b(+) His•Tag, S•Tag 70791-3
pET GST Fusion System 41 pET-41a–c(+) His•Tag, GST•Tag™, 70559-3
pET GST Fusion System 42 pET-42a–c(+)
S•Tag
His•Tag, GST•Tag,
70560-3
70564-3
7
pET NusA Fusion System 43.1 pET-43a–c(+) His•Tag, HSV•Tag®, 70942-3
System 43.1 plus Competent Cells Nus•Tag™, S•Tag 70943-3

8
inNovations Nos. 1, 3, 7–9, 11–13
9
10
11
Appendices
Indices

95
Novagen
Protein Expression
1 Fusion Tag Affinity Purification Products

Complete selection of affinity resins and companion products for tag-based purification of fusion proteins
pET System purification reagents and kits based on vector fusion tags. Note that CBIND™ Protein Purification for additional information.
are available separately to provide maximum purification reagents are included in the relevant Purification conditions are given in the
flexibility in choosing a purification strategy pET Expression Systems. Please see Chapter 9, Appendix.
2 Fusion Tag Description Product Size Cat. No.
GST•Tag™ Cross-linked agarose, 50% slurry in 20% ethanol, capacity 5–8 mg/ml settled resin GST•Bind™ Resin 10 ml 70541-3
50 ml 70541-4
Magnetic agarose beads derivatised with glutathione, capacity up to 2 mg/ml settled beads GST•Mag™ 2 × 1 ml 71084-3
Agarose Beads 10 × 1 ml 71084-4
3 Kit contains all buffers necessary for purification using GST•Bind Resin GST•Bind Buffer Kit 70534-3
Kit contains 10 ml GST•Bind Resin, Buffer Kit, BugBuster™ Reagent, Benzonase® BugBuster™ GST•Bind
Nuclease, chromatography columns Purification Kit 70794-3
Kit contains GST•Mag Agarose Beads, PopCulture™ Reagent, rLysozyme™ Solution, and PopCulture™ GST•Mag
buffers. Purification Kit 71113-3
4 Kit contains GST•Mag Agarose Beads, PopCulture Reagent, Benzonase Nuclease,

rLysozyme™ Solution, buffers, and 96-well Culture and Collection Plates with Sealers
RoboPop™ GST•Mag
Purification Kit 71102-3
His•Tag® Cross-linked NTA agarose, 50% slurry in 30% ethanol, pre-charged with nickel, capacity Ni-NTA His•Bind® Resin 10 ml 70666-3
5–10 mg/ml settled resin 25 ml 70666-4
100 ml 70666-5
5 Cross-linked NTA agarose, 50% slurry in 30% ethanol, pre-charged with nickel, capacity
5–10 mg/ml settled resin, high flow rates and pressures
Ni-NTA His•Bind
Superflow
10 ml
25 ml
100 ml
70691-3
70691-4
70691-5
Cross-linked IDA agarose, 50% slurry in 20% ethanol, provided as uncharged resin; must be His•Bind Resin 10 ml 69670-3
charged with nickel prior to use, capacity 8 mg/ml settled resin 50 ml 69670-4
100 ml 69670-5
Pre-packed columns containing 1.25 ml His•Bind Resin; pre-charged with nickel, capacity His•Bind Columns pkg/5 70971-3
6 10 mg/run pkg/25 70971-4
IDA cellulose, pre-charged with nickel, provided as dry resin, capacity ~2.5 mg/g resin His•Bind Quick Resin bulk 70694
Pre-packed, pre-charged cartridges designed for quick small-scale purification (~0.5 mg His•Bind Quick pkg/10 70155-3
protein/cartridge) 300 Cartridges pkg/50 70155-4
Pre-packed, pre-charged cartridges designed for quick small-scale purification (~2 mg His•Bind Quick pkg/10 70156-3
7 protein/cartridge)
Pre-packed, pre-charged columns designed for use with Novagen’s Vacuum Manifold
900 Cartridges
His•Bind Quick
pkg/50
pkg/12
70156-4
70159-3
(~5 mg protein/column) Columns pkg/60 70159-4
IDA tentacle Fractogel, suspension in 20% ethanol, 150 mM NaCl, provided as uncharged His•Bind Fractogel® (M) 25 ml 70693-3
resin; capacity > 10 mg/ml settled resin, high pressure compatible (40–90 µM)
IDA tentacle Fractogel, suspension in 20% ethanol, 150 mM NaCl, provided as uncharged His•Bind Fractogel (S) 25 ml 70692-3
8 resin; capacity > 10 mg/ml settled resin, high pressure compatible (20–40 µM)
Magnetic cross-linked IDA agarose, pre-charged with nickel, capacity 5 mg/ml settled beads His•Mag™ 2 × 1 ml 71002-3
Agarose Beads 10 × 1 ml 71002-4
Kit contains all buffers necessary for purification using His•Bind Resin under native His•Bind Buffer Kit 69755-3
conditions
9 Kit contains all buffers necessary for purification using His•Bind Quick Cartridges and
Columns under native conditions
His•Bind Quick
Buffer Kit 70665-3
Kit contains all buffers necessary for purification using Ni-NTA His•Bind Resins under native Ni-NTA Buffer Kit 70899-3
conditions
Kit contains 10 ml His•Bind Resin, Buffer Kit, and chromatography columns His•Bind Purification Kit 70239-3
Kit contains 10 ml Ni-NTA His•Bind Resin, BugBuster Reagent, Benzonase Nuclease, chro- BugBuster Ni-NTA
10 matography columns His•Bind Purification Kit 70751-3
Kit contains 10 ml His•Bind Resin, Buffer Kit, BugBuster Reagent, Benzonase Nuclease, BugBuster His•Bind
chromatography columns Purification Kit 70793-3
Kit contains His•Mag™ Agarose Beads, PopCulture Reagent, Benzonase Nuclease, PopCulture His•Mag
rLysozyme Solution, and buffers Purification Kit 71114-3
11 Kit contains His•Mag Agarose Beads, PopCulture Reagent, rLysozyme Solution, buffers, and
96-well Culture and Collection Plates with Sealers
RoboPop His•Mag
Purification Kit 71103-3
S•Tag™ Kit contains S-protein Agarose, Biotinylated Thrombin, spin filters, and all buffers necessary S•Tag™ Thrombin
for purification Purification Kit 69232-3
Appendices
Kit contains S-protein Agarose, Recombinant Enterokinase, spin filters, and all buffers neces- S•Tag rEK
Indices
sary for purification Purification Kit 69065-3
T7•Tag® Kit contains T7•Tag Antibody Agarose, columns, and all buffers necessary for purification T7•Tag® Purification Kit 69025-3

Novagen
Protein Expression
pET Expression Systems 3, 9, 11, 17b, and 17xb 1

Classic T7•Tag pET vectors
The pET-3, 9, and 11 vector series are origi- Features Product Size Cat. No.
nal, basic pET plasmids constructed by Studier • Choice of “plain” T7 (pET-3a–d, 9a–d, 17b, pET Expression System 3 69409-3
and colleagues (1), and are the precursors of 17xb) or T7lac (pET-11 series) promoters System plus Competent Cells 69410-3
many subsequent pET vectors developed by
Novagen. These vectors offer a single BamH I
• Ampicillin or kanamycin resistance pET-3a DNA 10 µg 69418-3 2
• pET-17b and 17xb contain dual BstX I sites, pET-3b DNA 10 µg 69419-3
cloning site in three reading frames for produc-
which enable efficient cloning using an
ing proteins fused with a non-cleavable N-termi- pET-3c DNA 10 µg 69420-3
asymmetric linker (2)
nal T7•Tag® epitope. Unfused proteins can be
• pET-17xb carries the extended 260 aa T7 pET-3d DNA 10 µg 69421-3
produced by using the Nde I cloning site in the
“a”, “b”, and “c” versions, or the Nco I site in the gene 10 fusion sequence, which includes the
11 aa T7•Tag epitope; larger fusion tag
pET Expression System 9
System plus Competent Cells
69415-3
69416-3
3
“d” version. The pET-17b and 17xb vectors carry
a multiple cloning site in one frame. stabilizes small target polypeptides pET-9a DNA 10 µg 69431-3
1. Studier, F. W., Rosenberg, A. H., Dunn, J. J. and pET-9b DNA 10 µg 69432-3
Dubendorff, J. W. (1990) Methods Enzymol. 185,
pET-9c DNA 10 µg 69433-3
2.
60–89.
Seed, B. (1987) Nature 329, 840. pET-9d DNA 10 µg 69434-3 4
pET Expression System 11 69405-3
System plus Competent Cells 69406-3
pET-11a DNA 10 µg 69436-3
la c l pET-11a-d pET-11b DNA
pET-11c DNA
10 µg
10 µg
69437-3
69438-3
5
T7lac pET-11d DNA 10 µg 69439-3
Nde I (Nco I in “d”)
T7•Tag pET System 17b 69726-3
BamH I System plus Competent Cells 69731-3
Bpu1102 I
T7 terminator pET-17b DNA 10 µg 69663-3 6
ori
pET System 17xb 69727-3

pET-17xb DNA 10 µg 69664-3
Ap
pET-3a-d pET-17b pET-17xb

Additional Information Available 7
pET-9a-d
T7 T7 T7 Vector Cloning Region Sequences Appendix
Nde I (Nco I in “d”) Nde I Nde I Vector maps and sequences www.novagen.com
T7•Tag T7•Tag T7•Tag (260 aa) Patent and Licensing Information p. 278
8
BamH I Hind III Sac II
Bpu1102 I Kpn I Hind III
o ri
T7 terminator Sac I Kpn I

BamH I Sac I
Spe I BamH I
BstX I Spe I
EcoR I BstX I
EcoR V EcoR I
BstX I EcoR V
Ap Not I BstX I
Kan Ap
9
Xho I Not I
pET-9a-d pET-3a-d Bpu1102 I Xho I
K an pET-17b T7 terminator Bpu1102 I
pET-17xb T7 terminator
10
11
Appendices
Indices

97
Novagen
Protein Expression
1 pET Expression Systems 14b, 15b, 16b, and 19b

Basic cleavable N-terminal His•Tag fusion vectors
The pET-14b, 15b, 16b, and 19b vectors are Features Product Size Cat. No.
designed for producing target proteins that con- • Choice of “plain” T7 (pET-14b) or T7lac pET Expression System 14b 70753-3
tain an N-terminal His•Tag® sequence and one of (pET-15b, 16b, 19b) promoters System plus Competent Cells 70754-3
2 three protease recognition sites. The His•Tag
sequence can be removed from purified target
• Ampicillin resistance pET-14b DNA 10 µg 69660-3
• Three cloning sites in single reading frame pET Expression System 15b 70755-3
proteins with thrombin (pET-14b, 15b), Factor
Xa (pET-16b), or enterokinase (pET-19b). • Unfused proteins can be expressed by System plus Competent Cells 70756-3
cloning into the Nco I site in these vectors pET-15b DNA 10 µg 69661-3
pET Expression System 16b 70757-3
3 pET-14b System plus Competent Cells 70758-3
pET-16b DNA 10 µg 69662-3
T7
Nco I System plus Competent Cells 70760-3
His•Tag
thrombin site pET-19b DNA 10 µg 69677-3
4 Nde I
o ri
Xho I
BamH I
Bpu1102 I
T7 terminator Additional Information Available
Ap Vector Cloning Region Sequences Appendix
5 la c l pET-15b pET-16b pET-19b

Patent and Licensing Information
www.novagen.com
p. 278
T7lac T7lac T7lac
Nco I Nco I Nco I
6 His•Tag
thrombin site
Nde I
His•Tag
Factor Xa site
Nde I
His•Tag
Ek site
Nde I
Xho I Xho I Xho I
BamH I BamH I BamH I
Bpu1102 I Bpu1102 I Bpu1102 I
ori
T7 terminator T7 terminator T7 terminator
7 Ap
pET-21(+), 23(+), and 24(+) Transcription Vectors

Expression of sequences carrying their own translation signals
8 The pET-21(+), 23(+), and 24(+) vectors are Features Product Size Cat. No.
“transcription” vectors that express RNA under • Choice of “plain” T7 [pET-23(+)] or T7lac pET-21(+) DNA 10 µg 69770-3
the control of T7 RNA polymerase, but do not [pET-21(+), 24(+)] promoters
provide bacterial translation signals. They are pET-23(+) DNA 10 µg 69771-3
• Ampicillin or kanamycin resistance
useful for expressing proteins from target genes pET-24(+) DNA 10 µg 69772-3
9 that contain their own bacterial ribosome bind-
ing site and ATG start codon.
• Multiple cloning sites followed by the
His•Tag coding sequence for optional
production of C-terminal His•Tag fusion Additional Information Available
proteins pET System Manual TB055
pET-21(+) pET-23(+) Vector Cloning Region Sequences Appendix
10 la c l
pET-24(+)
T7lac T7
www.novagen.com
p. 278
BamH I BamH I
EcoR I EcoR I
Sac I Sac I
Sal I Hinc II
Hind III Sal I
Not I Hind III
11 Eag I Not I
ri g i n
ri g i n
Xho I Eag I
ori
His•Tag Xho I
f1 o
f1 o
Bpu1102 I His•Tag
ori
T7 terminator Bpu1102 I
T7 terminator
Appendices
Indices
Ap Ap
Kan Ap
pET-24(+) pET-21(+)
K an

Novagen
Protein Expression
pET Expression Systems 12, 20b, 22b, 25b, 26b, and 27b 1
Signal sequence fusion vectors for potential export of target proteins to the periplasm
The pET-12a–c, 20b(+), 22b(+), 25b(+), Features Product Size Cat. No.
26b(+), and 27b(+) vectors are designed for • Choice of “plain” T7 or T7lac promoters for pET Expression System 12 69407-3
fusion of target proteins to signal sequences that control of expression levels System plus Competent Cells 69408-3
facilitate export into the periplasmic space. The
periplasm provides conditions that promote
• Ampicillin or kanamycin resistance pET-12a DNA 10 µg 69440-3 2
• Three reading frames available with the pET- pET-12b DNA 10 µg 69441-3
proper folding and disulfide bond formation,
12a–c series; others are in the “b” frame
which may enhance the solubility and activity of pET-12c DNA 10 µg 69442-3
certain target proteins. The signal sequence is • Optional C-terminal His•Tag® or HSV•Tag®/
His•Tag® combination (except pET 12a–c) pET Expression System 20b 70761-3
often cleaved by signal peptidase concomitant System plus Competent Cells 70762-3
with export. Other pET vectors with signal • Unfused proteins can be expressed by
pET-20b(+) DNA 10 µg 69739-3
3
sequences include pET-37b(+), pET-38b(+), pET- cloning into the Nde I site in these vectors
39b(+), and pET-40b(+). pET Expression System 22b 70765-3
pET-22b(+) DNA 10 µg 69744-3
pET-20b(+)
pET-12a-c T7
70771-3
70772-3
4
Nde I
signal sequence pET-25b(+) DNA 10 µg 69753-3
Nco I
T7 EcoR V
BamH I
Nde I EcoR I System plus Competent Cells 70774-3
5
signal sequence Sac I
BamH I ri g i n Hinc II pET-26b(+) DNA 10 µg 69862-3
ori
o ri
Bpu1102 I Sal I
f1 o
T7 terminator Hind III

Not I
Eag I System plus Competent Cells 70776-3
Xho I
His•Tag pET-27b(+) DNA 10 µg 69863-3
Bpu1102 I
Ap Ap T7 terminator
Additional Information Available 6

pET-22b(+) pET-25b(+) inNovations Nos. 1, 3, 7–9, 11–13
pET-26b(+) pET-27b(+) Vector maps and sequences www.novagen.com
la c l T7lac T7lac Patent and Licensing Information p. 278
Nde I
signal sequence
Msc I
Nde I
signal sequence
Msc I
7
Nco I Nco I
BamH I BamH I
EcoR I EcoR I
Sac I Sac I
ri g i n
Sal I Sal I
Hind III Hind III
f1 o
Not I Not I
8
ori
Eag I Eag I
Xho I Xho I
His•Tag Nhe I
Bpu1102 I HSV•Tag
T7 terminator His•Tag
Ap Bpu1102 I
Kan Ap T7 terminator
K an pET-26b(+) pET-22b(+)
pET-27b(+) pET-25b(+)
10
11
Appendices
Indices

99
Novagen
Protein Expression
1 pET Expression Systems 21, 23, and 24 Product Size Cat. No.
®
Versatile T7•Tag fusion vectors including optional C-terminal His•Tag sequence ® pET Expression System 21 70763-3
The pET-21, 23, and 24 series of vectors are “a”, “b”, and “c” versions, or the Nco I site in the pET-21a(+) DNA 10 µg 69740-3
designed for producing target proteins fused “d” version. pET-21b(+) DNA 10 µg 69741-3
with a non-cleavable N-terminal T7•Tag® epi- Features pET-21c(+) DNA 10 µg 69742-3
2 tope, with the option to include a C-terminal
His•Tag® sequence. C-terminal His•Tag fusions
• Choice of “plain” T7 [pET-23a–d(+)] or T7lac
pET-21d(+) DNA 10 µg 69743-3
[pET-21a–d(+), 24a–d(+)] promoters
are created by cloning inserts that are in the cor- pET Expression System 23 70767-3
rect reading frame and lack a stop codon. • Ampicillin or kanamycin resistance System plus Competent Cells 70768-3
Proteins having their native N-terminus can be • Multiple cloning sites in all three reading
pET-23a(+) DNA 10 µg 69745-3
produced by using the Nde I cloning site in the frames
3 pET-23b(+) DNA 10 µg 69746-3
pET-23c(+) DNA 10 µg 69747-3
pET-23a-d(+) pET-21a-d(+) pET-23d(+) DNA 10 µg 69748-3
pET-24a-d(+)
T7 la c l
pET Expression System 24 70769-3
T7lac
4 Nde I (Nco I in “d”)
T7•Tag
BamH I
Nde I (Nco I in “d”)
T7•Tag
BamH I
pET-24a(+) DNA 10 µg 69749-3
EcoR I
Sac I
EcoR I pET-24b(+) DNA 10 µg 69750-3
Sac I
Hinc II Sal I
Sal I Hind III
pET-24c(+) DNA 10 µg 69751-3
ri g i n
ri g i n
Hind III Not I
ori
Not I Eag I pET-24d(+) DNA 10 µg 69752-3

f1 o
Eag I
f1 o
5
Xho I
Xho I
ori
His•Tag
His•Tag Bpu1102 I
Bpu1102 I
T7 terminator
T7 terminator Additional Information Available
Ap pET System Manual TB055
Ap
Kan Ap inNovations Nos. 1, 3, 7–9, 11–13
pET-24a-d(+) pET-21a-d(+) Vector Cloning Region Sequences Appendix
K an
6 Patent and Licensing Information p. 278
pET Expression Systems 28, 29, and 30 Product Size Cat. No.
Cleavable N-terminal His•Tag, T7•Tag, and S•Tag fusion vectors pET Expression System 28 70777-3
7 The pET-28, 29, and 30 series of vectors are two versions as a linearized vector prepared for pET-28a(+) DNA 10 µg 69864-3
designed for generating target proteins fused ligation-independent cloning (LIC) of PCR prod- pET-28b(+) DNA 10 µg 69865-3
with cleavable N-terminal His•Tag/T7•Tag (pET- ucts. With these versions, all vector-encoded N-
28), His•Tag/S•Tag™ (pET-30), or S•Tag only terminal fusion sequences can be removed from
pET-28c(+) DNA 10 µg 69866-3
(pET-29) sequences, with the option to include a purified proteins with either enterokinase pET Expression System 29 70779-3
8 C-terminal His•Tag sequence. C-terminal
His•Tag fusions are created by cloning inserts
(Ek/LIC) or Factor Xa (Xa/LIC).
Features
pET-29a(+) DNA 10 µg
70780-3
69871-3
that are in the correct reading frame and lack a pET-29b(+) DNA 10 µg 69872-3
• T7lac promoter
stop codon. Proteins having their native N-termi-
nus can be produced by using the Nde I (pET-29, • Kanamycin resistance pET-29c(+) DNA 10 µg 69873-3
30) or Nco I site (pET-28). The vectors encode • Multiple cloning sites in all three reading pET Expression System 30 70781-3
9 protease recognition sites for removal of one or frames System plus Competent Cells 70782-3
both N-terminal tags. pET-30 is also available in pET-30a(+) DNA 10 µg 69909-3
pET-30b(+) DNA 10 µg 69910-3
pET-28a-c(+) pET-29a-c(+) pET-30a-c(+)
pET-30c(+) DNA 10 µg 69911-3
T7lac T7lac T7lac
10 la c l
Nco I
His•Tag
Nde I
S•Tag
Nde I
His•Tag
pET-30 Ek/LIC
Vector Kit 20 rxn 69077-3
thrombin site Bgl II thrombin site
Nde I Kpn I S•Tag pET-30 Ek/LIC Vector 1 µg 69024-3
T7•Tag thrombin site Bgl II (linearized vector)
BamH I Nco I Kpn I
EcoR I EcoR V Ek site pET-30 Xa/LIC
Sac I BamH I Nco I
EcoR I EcoR V Vector Kit 20 rxn 70073-3
ri g i n
Sal I
11 Hind III
Not I
Sac I
Sal I
BamH I
EcoR I pET-30 Xa/LIC Vector 1 µg 70022-3
f1 o
Eag I Hind III Sac I (linearized vector)

o ri
Xho I Not I Sal I

His•Tag Eag I Hind III
Bpu1102 I Xho I Not I
T7 terminator His•Tag Eag I
K an Bpu1102 I Xho I
Appendices
T7 terminator His•Tag
Indices
Bpu1102 I
T7 terminator inNovations Nos. 1, 3, 7–9, 11–13

Novagen
Protein Expression
pET Peptide Expression System 31b Product Size Cat. No.

1
High yield bioproduction of peptides and small proteins pET Expression System 31b 70783-3
ence of T7 RNA polymerase provided by the pET-31b(+) DNA 10 µg 69952-3
la c l pET-31b(+)
recA–, protease deficient BLR(DE3)pLysS host pET-31b(+) DNA, AlwN I 40 rxn 69954-3
T7lac
(included in the Expression System). Purified digested, dephosphorylated
Nde I
Nsi I
KSI
fusion proteins are cleaved with CNBr to release
the His•Tag peptide, insoluble KSI, and
Components:
• 2 µg AlwN I digested, dephosphorylated
2
AlwN I
Ava I monomer peptide units each terminating with a pET-31b(+) DNA
Xho I
ri g i n
His•Tag homoserine lactone. The purified peptide can be • 2 pmol AlwN I Control Insert
Bpu1102 I • 0.2 ml Induction Control glycerol stock
easily converted to an amide or homoserine, or
f1 o
T7 terminator
ori
reacted with other compounds, such as analogs

of fluorescein and biotin, to produce C-terminal
3
derivatized peptides. The system has been used
Ap pET System Manual TB055
to make biologically active peptides of 14–67 aa
pET Peptide Expression System 31b TB104
at yields of > 50 mg purified peptide per liter of Vector maps and sequences www.novagen.com
The pET Peptide Expression System 31b is pET-31b(+) Cloning Reqion Sequence Appendix
culture (1, 2).
designed for expression and purification of pep- inNovations No. 2
tides and small proteins up to 100 amino acids in
A supplementary kit (Cat. No. 69954-3) is
available that contains the AlwN I digested,
Patent and Licensing Information p. 278 4
length. In pET-31b(+), peptide coding sequences
dephosphorylated vector ready to accept appro-
are placed downstream of a 125 amino acid (aa)
priate inserts containing ...ATG-3' and 3'-TAC...
ketosteroid isomerase (KSI) gene and upstream
overhangs on the top and bottom strands,
of a His•Tag® sequence. When induced, a
respectively. A Control Insert is provided to veri-
hydrophobic KSI-peptide-His•Tag fusion protein
is produced in large amounts as insoluble aggre-
fy performance and ensure optimal cloning effi-
ciency.
5
gates in the E. coli cytoplasm, such that target
sequences are protected from degradation. Features
Fusion proteins can be solubilized and purified • High yield expression of peptides and small
under denaturing conditions using a His•Bind® proteins
affinity matrix. Typical yields are in the range of
50 mg purified protein per liter of culture.
• His•Tag sequence facilitates protein
purification
6
Use of the unique AlwN I cloning site enables • Prepared vector facilitates cloning with
unidirectional insertion of coding sequences minimal background
immediately adjacent to a methionine residue.
• Protease-deficient host strain facilitates
For high yield production, small (10–25 aa) pep-
tide coding sequences can be cloned as tandem
production of full-length proteins
7
repeats interspersed with single methionine 1. Kuliopulos, A. and Walsh, C. T. (1994) J. Am. Chem.
residues (1, 2). The KSI-peptide-His•Tag fusion Soc. 116, 4599–4607.
protein is expressed at high levels in the pres- 2. Kuliopulos, A. (1994) inNovations 2, 4–7.
Cloning, expression and purification of short peptides using AlwN I digested pET-31b(+)
8
Met Met
5'P- coding sequence ATG-3'
3'-TAC -5'P
anneal
ligate
Met
TAC
coding sequence ATG
TAC
Met
coding sequence ATG
TAC coding sequence
n
Met
ATG
Met
9
T7
+
GCA TGC CAG ATG CTG CTC GAG CAC TGA
KSI CGT ACG GTC ()
TAC GAC GAG CTC GTG ACT
6
AlwN I cut, dephosphorylated pET-31b(+) lac
T7lac ▼
ligate
▼ ▼ ▼
10
KSI coding sequence coding sequencen coding sequence His•Tag ®
expression
His•Bind® chromatography
CNBr ▼ cleavage
KSI
(insoluble carrier protein)

His•Tag® peptide
peptide
peptide
peptide
11
Appendices
Indices

101
Novagen
Protein Expression
1 pET Trx Fusion System 32 Product Size Cat. No.
Production of soluble, active target proteins in E. coli pET Trx Fusion System 32 70785-3
pET-32a-c(+) cytoplasmic disulfide metabolism (see figure pET-32a(+) DNA 10 µg 69015-3
T7lac below). These strains therefore promote even pET-32b(+) DNA 10 µg 69016-3
la c l higher levels of disulfide bonding in the E. coli
Trx•Tag
pET-32c(+) DNA 10 µg 69017-3
2 Msc I
His•Tag
thrombin site
cytoplasm (5). Expression of pET-32 constructs
in a trxB/gor host may yield the maximum levels pET-32 Ek/LIC
S•Tag
Bgl II of soluble, active, properly folded target protein. Vector Kit 20 rxn 69076-3
Kpn I
Ek site pET-32 is also available in two additional ver- pET-32 Ek/LIC Vector 1 µg 69023-3
Nco I (linearized vector)
sions prepared for ligation-independent cloning
ri g i n
EcoR V
BamH I (LIC) of PCR products. With these versions, all pET-32 Xa/LIC
f1 o
3 EcoR I
ori
Sac I vector-encoded N-terminal fusion sequences can

Sal I
Hind III be removed from purified proteins with either pET-32 Xa/LIC Vector 1 µg 70023-3
Not I (linearized vector)
Eag I enterokinase (Ek/LIC) or Factor Xa (Xa/LIC).
Ap Xho I
Features
AD494 0.4 ml 69033-3
Ava I
His•Tag Competent Cells 1 ml 69033-4
Bpu1102 I • Fusion to thioredoxin for increased protein
4 T7 terminator
solubility
AD494(DE3)
Competent Cells
0.4 ml
1 ml
69013-3
69013-4
• Protease cleavage sites for complete fusion
The popular pET Trx Fusion System 32 is AD494(DE3)pLysS 0.4 ml 69014-3
tag removal Competent Cells 1 ml 69014-4
designed for cloning and high-level expression of
polypeptide sequences fused with the 109 aa • Compatible with AD494, BL21trxB, Origami, BL21trxB(DE3) 0.4 ml 70508-3
Trx•Tag™ thioredoxin protein. Many proteins and Rosetta-gami host strains to promote Competent Cells 1 ml 70508-4
5 that are normally produced in an insoluble form
in E. coli tend to become more soluble when •
proper folding of expressed protein
His•Tag® sequence facilitates protein
BL21trxB(DE3)pLysS
Competent Cells
0.4 ml
1 ml
70509-3
70509-4
fused with the Trx•Tag sequence (1, 2). The purification Origami™ 0.4 ml 70626-3
pET-32 vectors are also compatible with trxB • S•Tag™ sequence for sensitive protein Competent Cells 1 ml 70626-4
mutant hosts AD494 and BL21trxB, and with the quantification, non-radioactive detection, and Origami(DE3) 0.4 ml 70627-3
trxB/gor mutant Origami™, Origami B, and
6 Rosetta-gami™ strains. The thioredoxin reduc-
affinity purification Competent Cells
Origami(DE3)pLysS
1 ml
0.4 ml
70627-4
70628-3
tase (trxB) mutation has been shown to allow 1. LaVallie, E. R., DiBlasio, E. A., Kovacic, S., Grant, K.
L., Schendel, P. F. and McCoy, J. M. (1993) Competent Cells 1 ml 70628-4
the formation of disulfide bonds in the E. coli Bio/Technology 11, 187–193. Origami(DE3) Singles™ 11 rxn 70630-3
cytoplasm (3). The thioredoxin fusion tag
2. Novy, R., Berg, J., Yaeger, K., and Mierendorf, R. Competent Cells 22 rxn 70630-4
expressed from pET-32 vectors not only (1995) inNovations 3, 7–9.
Origami(DE3)pLysS
7 enhances the solubility of many target proteins,
but appears to catalyze the formation of disul-
3. Derman, A. I., Prinz, W. A., Belin, D., and Beckwith,
J. (1993) Science 262, 1744–1747.
Singles Competent
Cells
11 rxn
22 rxn
70631-3
70631-4
fide bonds in the cytoplasm of trxB mutants (4). 4. Stewart E. J., Aslund, F., and Beckwith, J. (1998)
EMBO J. 17, 5543–5550. Rosetta-gami™ 0.4 ml 71054-3
The Origami and Rosetta-gami hosts possess an Competent Cells 1 ml 71054-4
additional mutation in the glutathione reductase 5. Prinz, W. A., Aslund, F., Holmgren, A., and
(gor) gene, which is the other central enzyme in Beckwith, J. (1997) J. Biol. Chem. 272, Rosetta-gami(DE3) 0.4 ml 71055-3
8 15661–15667. Competent Cells
Rosetta-gami(DE3)pLysS
1 ml
0.4 ml
71055-4
71057-3
Competent Cells 1 ml 71057-4
Thioredoxin System
thioredoxin thioredoxin pET System Manual TB055
9 reductase
(trxB)
(trxA) Xa/LIC Vector Kits Protocol
Ek/LIC Vector Kits Protocol
TB184
TB163
NADPH Protein inNovations No. 3
glutathione Patent and Licensing Information p. 278
glutathione (gshA gshB)
reductase
10 (gor)
glutaredoxin 1-3
(grxA grxB grxC)
Glutaredoxin System
Pathways that determine the thiol-disulfide balance in the E. coli cytoplasm
11 Strains possessing mutant trxB or trxB and gor genes are permissive for the formation of disulfide bonds in cytoplasmically expressed target pro-
teins. From reference (5); printed with permission.
Appendices
Indices

Novagen
Protein Expression
pET Expression System 33b Product Size Cat. No.

1
Unique system for production of target proteins suitable for site-specific P-labeling 32 pET Expression System 33b 70787-3
pET-33b(+) PKA recognition sequence (ArgArgAlaSerVal), pET-33b(+) DNA 10 µl 69054-3
and T7•Tag® peptides. Fusion proteins
la c l T7lac
expressed from this plasmid can be affinity puri-
Nco I
His•Tag
thrombin site
fied with either His•Bind® resins or T7•Tag
Antibody Agarose, and then labeled in vitro in a pET System Manual TB055 2
kinase site PKAce Kit protocol TB231
Nde I simple reaction with 32P-g-ATP and purified PKA inNovations No. 4a
T7•Tag
BamH I
(available in the PKAce™ Kit). Proteins can be pET-33b(+) Cloning Region Sequence Appendix
EcoR I labeled to at least 105 cpm/µg in 10 minutes, and Vector maps and sequences www.novagen.com
ri g i n
Sac I Patent and Licensing Information p. 278

Sal I can be used directly as sensitive and specific
3
f1 o
Hind III
o ri
Not I probes for protein:protein interactions (2). A

Eag I
Xho I powerful method that uses such probes for rapid
His•Tag mapping of interacting protein domains is illus-
K an Bpu1102 I
T7 terminator trated below (3, 4).
1. Blanar, M. A. and Rutter, W. J. (1992) Science 256,
The pET-33b(+) vector is a unique expres-
sion plasmid that encodes a 5 aa recognition
2.
1014–1018.
de Arruda, M., and Burgess, R. R. (1995)
4
sequence for the catalytic subunit of cAMP- inNovations 4a, 7–8.
dependent protein kinase (protein kinase A; 3. Burgess, R. R., Arthur, T. M., and Pietz, B. C. (1998)
PKA) from heart muscle (1, 2). The vector- Cold Spring Harb Symp Quant Biol 63, 277–287.
encoded fusion sequences available on pET- 4. Arthur, T. M., and Burgess, R. R. (1998) J. Biol.
33b(+) include His•Tag®, thrombin cleavage site, Chem. 273, 31381–31387.
5
Probe Protein
Target Protein
Interaction domain
Denature
6
Cleave, purify fragments on
Ni-NTA His•Bind Resin
SDS-PAGE, blot, refold
Incubate with 32 P-labeled Probe Protein Interaction domain mapping with 32P-labeled protein expressed
from pET-33b(+)
7
32P 32P
The ability to label recombinant proteins expressed from pET-33b(+) with 32P has been used in the development of
a method for studying protein:protein interactions. Two recombinant clones of a target protein, which contain either
His6 His6 an N-terminal or C-terminal His•Tag (His6) sequence, are constructed in appropriate pET vectors. The unlabeled tar-
32P 32P
get proteins are expressed and subjected to partial chemical cleavage, followed by purification of the resulting frag-
His6
32P
His6 ments by metal chelation chromatography under denaturing conditions. The resulting ordered fragments are sepa-
rated on SDS-PAGE, blotted to nitrocellulose, briefly refolded on the membrane, and probed with 32P-labeled protein
8
expressed in pET-33b(+) in an “Ordered Fragment Ladder Far-Western” (3, 4). Because the fragments are all derived
His6 His6
from one end of the target protein, the fragment at which the probe protein no longer binds defines the interacting
domain. Figure courtesy of R. Burgess, University of Wisconsin.
His6 His6
N-terminal Ladder C-terminal Ladder 9
PKAce™ Kit Product Size Cat. No.
Convenient kit for site-specific 32P-labeling of target proteins PKAce™ Kit 20 rxn 70510-3 10
The PKAce™ Kit is designed for in vitro tions (1). The PKAce Kit includes a highly puri-
labeling of proteins in a simple reaction with fied preparation of bovine heart PKA, optimized
32 PKAce Kit Protocol TB231
P-g-ATP and purified Protein Kinase A (PKA). reaction buffer, and PKA Control Protein for ver-
inNovations No. 4a
Proteins can be labeled to at least 105 cpm/µg in ification of kit performance.
10 minutes, and can be used directly as sensitive
1. de Arruda, M. and Burgess, R. R. (1995)
11
and specific probes for protein:protein interac- inNovations 4a, 7–8.
Appendices
Indices

103
Novagen
Protein Expression
1 pET CBD Fusion Systems 34b–38b

Cellulose binding domain technology for immobilization and purification of target proteins
The pET CBD Fusion Systems 34b–38b Configurations Product Cat. No.
include vectors that enable the expression of Three different CBD•Tag vector configura- pET CBD Fusion System 34b 70110-3
target proteins fused with cellulose binding tions are available, which allow for cytoplasmic System plus Competent Cells 70112-3
2 domain (CBD•Tag™) sequences. The CBD•Tag
peptides offer significant advantages for a vari-
[pET-34b(+), pET-35b(+)] or potential periplas-
mic [pET-36b(+), pET-37b(+), and pET-38b(+)]
pET CBD Fusion System 35b 70111-3
ety of applications, but are especially suited for localization of expressed proteins.
protein immobilization and bioaffinity separa-
All CBD•Tag vectors except pET-38b(+) are System plus Competent Cells 70148-3
tion. also available as linearized vectors prepared for
ligation-independent cloning (LIC) of PCR prod-
3 Features
• Use of low cost, biocompatible cellulose ucts. With these versions, all vector-encoded N-
pET CBD Fusion System 38b
70150-3
70151-3
supports with inherent low non-specific terminal fusion sequences can be removed from
binding and no diffusion within the matrix purified proteins with either enterokinase
(Ek/LIC) or Factor Xa (Xa/LIC). Components:
(unlike agarose)
• pET vector DNA, 10 µg
• High capacity and specific retention without 1. Novy, R., Yaeger, K. and Miller, S. (1997) • Host bacterial strains BL21, BL21(DE3), and
4 the need for covalent modification; no
2.
inNovations 7, 4–7.
Ong, E., Gilkes, N. R., Miller, R. C., Jr., Warren, R. A.
leaching, no metals or inorganics required • Induction control clone, glycerol stock
J., and Kilburn, D. G. (1991) Enzyme Microb.
• Rapid binding kinetics enable processing of • CBIND™ 300 Cartridges, 5 each
Technol. 13, 59–65.
• CBIND 900 Cartridges, 5 each
large volumes or dilute solutions under a 3. Ramirez, C., Fung, J., Miller, R. C., Jr., Warren, R. A.
• CBIND Elute Reagent, 10 ml
wide range of conditions J., and Kilburn, D. G. (1993) Bio/Technology 11,
5 • Availability of vectors for secretion to enable

purification directly from media (1) 4.
1570–1573.
Tomme, P., Gilkes, N. R., Miller, R. C., Jr., Warren,
Product
pET-34b(+) DNA
Size
10 µg
Cat. No.
70102-3
R. A. J., and Kilburn, D. G. (1994) Protein
• Retention of functional activity of target Engineering 7, 117–123. pET-35b(+) DNA 10 µg 70103-3
proteins (2–4)
pET-36b(+) DNA 10 µg 70133-3
pET-37b(+) DNA 10 µg 70135-3
6 pET-34b(+) pET-36b(+) pET-38b(+) pET-38b(+) DNA 10 µg 70137-3
pET-35b(+) pET-37b(+) pET-34 Ek/LIC
T7lac T7lac T7lac Vector Kit 20 rxn 70114-3
la c l
CBDclos•Tag Nde I Nde I pET-35 Xa/LIC
SexA I signal sequence signal sequence
7 Sac II
thrombin site
Sca I
CBDcenA•Tag
SexA I
thrombin site
Nhe I
Nco I
EcoR V pET-36 Ek/LIC
Mun I Sca I BamH I
S•Tag Mun I EcoR I
Bgl II S•Tag UbaE I
Kpn I Bgl II BsrG I pET-37 Xa/LIC
ri g i n
Ek (34) or Xa (35) site Kpn I Sse8387 I Vector Kit 20 rxn 70153-3

BseR I Ek (36) or Xa (37) site Pst I
f1 o
Srf I BseR I Aat II

pET-34 Ek/LIC Vector 1 µg 70100-3
o ri
8 Nco I
EcoR V
BamH I
Srf I
Nco I
EcoR V
Hind III
Not I
Xho I
(linearized vector)
EcoR I BamH I thrombin site pET-35 Xa/LIC Vector 1 µg 70101-3

K an
Sac I EcoR I Ahd I (linearized vector)
Hind III Sac I Spe I
Not I Hind III CBDcex•Tag pET-36 Ek/LIC Vector 1 µg 70134-3
Eag I Not I His•Tag
Xho I
(linearized vector)
Eag I Avr II
His•Tag Xho I Bpu1102 I
pET-37 Xa/LIC Vector 1 µg 70136-3
9 Avr II
Bpu1102 I
T7 terminator
His•Tag
Avr II
Bpu1102 I
T7 terminator
(linearized vector)
T7 terminator

Notes: pET-34b(+) and pET-36b(+) contain an enterokinase cleavage site, whereas pET-35b(+) and pET-37b(+) contain a Factor Xa site. CBIND™ Kits Protocol TB189
The CBDcenA•Tag sequence in pET-36b(+) and pET-37b(+) encodes its own signal sequence for protein export.
10 Ek/LIC Vector Kits Protocol
Xa/LIC Vector Kits Protocol
inNovations
TB163
TB184
No. 7
11
Appendices
Indices

Novagen
Protein Expression
pET Dsb Fusion Systems 39b and 40b Product Size Cat. No.
1
Vectors for export and periplasmic folding of target proteins pET Dsb Fusion System 39b 70789-3
The pET Dsb Fusion Systems 39b and 40b • His•Tag® sequence facilitates protein pET-39b(+) DNA 10 µg 70090-3
are designed for cloning and high-level expres- purification pET Dsb Fusion System 40b 70791-3
sion of peptide sequences fused with the 208 aa • S•Tag™ sequence for sensitive protein System plus Competent Cells 70792-3
DsbA•Tag™ sequence [pET-39b(+)] or the 236
aa DsbC•Tag™ sequence [pET-40b(+)].
quantification, non-radioactive detection, and
efficient purification
pET-40b(+) DNA 10 µg 70091-3 2
DsbA and DsbC are periplasmic enzymes DsbA•Tag™ Primer 500 pmol 70177-3
that catalyze the formation and isomerization of 1. Rietsch, A., Belin, D., Martin, N. and Beckwith, J. DsbC•Tag™ Primer 500 pmol 70178-3
(1996) Proc. Natl. Acad. Sci. USA 93, 13048–13053.
disulfide bonds, respectively (1–5). In contrast to
2. Sone, M., Akiyama, Y., and Ito, K. (1997) J. Biol.
the pET-32 Trx•Tag™ series, which is designed
to increase solubility in the cytoplasm, the DsbA
3.
Chem. 272, 10349–10352.
Missiakas, D., Georgopoulas, C., and Raina, S.
3
and DsbC•Tag vectors enable potential periplas- (1994) EMBO J. 13, 2013–2020. Vector Cloning Region Sequences Appendix
mic localization and thus may enhance solubility Vector maps and sequences www.novagen.com
4. Zapun, A., Missiakas, D., Raina, S., and Creighton, T.
and proper folding in this non-reducing environ- E. (1995) Biochemistry 34, 5075–5089. Patent and Licensing Information p. 278
ment (6). 5. Raina, S., and Missiakas, D. (1997) Ann. Rev.
Features
• Fusion to DsbA or DsbC for export and
6.
Microbiol. 51, 179–202.
Collins-Racie, L. A., McColgan, J. M., Grant, K. L.,
4
DiBlasio-Smith, E. A., McCoy, J. M., and LaVallie, E.
increased protein solubility/folding in the R. (1995) Bio/Technology 13, 982–987.
periplasm
tag removal
5
pET-39b(+) pET-40b(+)
T7lac T7lac
la c l DsbA•Tag DsbC•Tag
Spe I
His•Tag
Sac II
thrombin site
Spe I
His•Tag
Sac II
thrombin site
6
Sca I Sca I
S•Tag Mun I
Nsp V S•Tag
Kpn I Nsp V
ri g i n
Ek site Bgl II
BseR I Kpn I
f1 o
Srf I Ek site
7
o ri
Nco I BseR I
BamH I Srf I
EcoR I Nco I
Sac I EcoR V
Sal I BamH I
K an Hind III EcoR I
Not I Sac I
Eag I Sal I
Xho I Hind III
His•Tag
Avr II
T7 terminator
Not I
Eag I
Xho I
His•Tag
8
Avr II
T7 terminator
10
11
Appendices
Indices

105
Novagen
Protein Expression
1 pET GST Fusion Systems 41 and 42 Product Size Cat. No.
Popular GST fusion tag in advanced pET vectors pET GST Fusion System 41 70559-3
pET-41a-c(+) In contrast to other commercially available pET-41a(+) DNA 10 µg 70556-3

pET-42a-c(+) GST-fusion vectors, the Xa (IleGluGlyArg, pET- pET-41b(+) DNA 10 µg 70557-3
T7lac 42 series) and enterokinase (AspAspAspAspLys,
pET-41c(+) DNA 10 µg 70558-3
2 la c l Nde I
GST•Tag
pET-41 series) proteolytic cleavage sites have
been engineered to allow 100% complete pET GST Fusion System 42 70564-3
Spe I
His•Tag removal of vector encoded sequences and the System plus Competent Cells 70565-3
Sac II
thrombin site generation of native proteins with their authentic pET-42a(+) DNA 10 µg 70561-3
Mun I
S•Tag
N-terminal residues. A version of pET-41 is avail- pET-42b(+) DNA 10 µg 70562-3
Bgl II able as a linearized vector prepared for ligation-
3
ri g i n
Kpn I
PinA I independent cloning (LIC) of PCR products. pET-42c(+) DNA 10 µg 70563-3
f1 o
Ek (41) or Xa (42) site

The His•Tag and S•Tag sequences enable pET-41 Ek/LIC
o ri
PshA I
Nco I
alternative protein detection and purification Vector Kit 20 rxn 71071-3
EcoR V
BamH I procedures to be performed: for example, when
EcoR I
K an BsrG I enhancing purity with a separate purification (linearized vector)
Stu I
GST•Bind™ Resin 10 ml 70541-3
4 Asc I
Sse8387 I
Pst I
method, or when purifying under denaturing
conditions. 50 ml 70541-4
Sac I
Sal I Features GST•Mag™ 2 × 1 ml 71084-3
Hind III Agarose Beads 10 × 1 ml 71084-4
Not I • Fusion to widely used GST•Tag sequence for
Eag I
high protein yield and increased solubility GST•Bind Buffer Kit 70534-3
Xho I
His•Tag
5 Avr II
Bpu1102 I
T7 terminator
tag removal
Components:
• 2 × 100 ml 10X GST Bind/Wash Buffer
• 1g Reduced Glutathione
• One step, gentle affinity purification with
• 40 ml 10X Glutathione Reconsitution
The pET-41a–c(+) and pET-42a–c(+) vectors GST•Bind™ Resins and kits Buffer
incorporate the schistosomal glutathione-S- • Quantification of target proteins with
transferase (GST; GST•Tag™) coding sequence Product Size Cat. No.
GST•Tag Assay Kit
6 as a fusion partner. The GST•Tag™ sequence
has been reported to enhance the production
• Sensitive detection on Western blots and GST•Tag™
Assay Kit 100 assays 70532-3
immunofluorescence with GST•Tag
and in some cases the solubility of its fusion Components:
Monoclonal Antibody
partners (1). When expressed in a soluble, prop- • 2 × 5 ml 10X GST•Tag Assay Buffer
erly folded form, GST•Tag fusion proteins can • His•Tag and S•Tag sequences also available
• 1.2 ml 100 mM CDNB
be purified with immobilized glutathione. Gentle for additional detection and purification
7 elution is achieved with buffers containing options
• 1g
• 50 µg
Reduced Glutathione
GST•Tag Standard
reduced glutathione. Quantification of soluble • All detection and purification options
compatible with BugBuster™ and Product Size Cat. No.
GST fusions is also possible by assaying the
transferase activity. The pET-41 and -42 series PopCulture™ Extraction Reagents GST•Tag 50 µg 71097-3
feature the powerful T7lac promoter, and Monoclonal Antibody 250 µg 71097-4
1. Smith, D. B. and Johnson, K. S. (1988) Gene 67,
8 encode the GST•Tag (220 aa), proteolytic sites,
His•Tag® (6 aa) and S•Tag™ (15 aa) sequences.
31–40.
GST•Bind Kits Protocol TB235
GST•Tag Assay Kit Protocol TB236
9 Vector maps and sequences
www.novagen.com
p. 278
10
11
Appendices
Indices

Novagen
Protein Expression
pET NusA Fusion System 43.1 Product Size Cat. No.

1
Production of soluble, active proteins in E. coli pET NusA Fusion System 43.1 70942-3
pET-43.1a-c(+) pET-43.1 vectors incorporate the Nus•Tag fusion pET-43.1a(+) DNA 10 µg 70939-3
T7lac partner and are also compatible with the trxB/gor pET-43.1b(+) DNA 10 µg 70940-3
la c l Nde I mutant Origami™, Origami B and Rosetta-
pET-43.1c(+) DNA 10 µg 70941-3
Nus•Tag
Spe I
His•Tag
gami™ strains, which facilitate disulfide bond
formation in the cytoplasm (2). Disulfide bonded pET-43.1 Ek/LIC
2
Sac II
S•Tag proteins may be obtained by employing the com- Vector Kit 20 rxn 71072-3
thrombin site
Sma I bination of the pET 43.1 vector and these pET-43.1 Ek/LIC Vector 1 µg 71070-3
Ek site (linearized vector)
trxB/gor host strains.
ri g i n
PshA I
Sac I A version of pET-43.1 is available as a lin- AD494(DE3) 0.4 ml 69013-3
3
f1 o
BamH I
earized vector prepared for ligation-independent Competent Cells 1 ml 69013-4
o ri
EcoR I
BsrG I
Asc I cloning (LIC) of PCR products. With this version, Origami™ 0.4 ml 70626-3
Sse8387 I
all vector-encoded N-terminal fusion sequences Competent Cells 1 ml 70626-4
Pst I
Ap Sal I
Kpn I
can be removed from purified proteins with Origami(DE3) 0.4 ml 70627-3
Hind III enterokinase. Competent Cells 1 ml 70627-4
Not I
Eag I
Pml I
HSV•Tag
Features
• N-terminal Nus•Tag fusion partner,
Origami(DE3)pLysS
Competent Cells
0.4 ml
1 ml
70628-3
70628-4 4
Xho I
His•Tag systematically identified for high solubility Origami(DE3) Singles™ 11 rxn 70630-3
Pac I
Avr II
Competent Cells 22 rxn 70630-4
T7 terminator
• Upstream His•Tag®, S•Tag™ and optional
C-terminal HSV•Tag® and His•Tag fusion Origami(DE3)pLysS 11 rxn 70631-3
Singles Competent Cells 22 rxn 70631-4
The pET NusA Fusion System 43.1 is
designed for cloning and high-level expression of •
tags
Thrombin and enterokinase cleavage sites
Origami B
Competent Cells
0.4 ml
1 ml
70836-3
70836-4
5
polypeptide sequences fused with the 495 aa • Multiple cloning sites in all reading frames
Origami B(DE3) 0.4 ml 70837-3
NusA (Nus•Tag™) protein. The NusA protein • Compatible with Origami, Origami B, and Competent Cells 1 ml 70837-4
has been identified as having the highest poten- Rosetta-gami host strains to promote proper
Origami B(DE3)pLysS 0.4 ml 70839-3
tial for solubility when a data base of more than
4000 E. coli proteins was subjected to solubility
folding of expressed protein Competent Cells
Rosetta-gami™
1 ml
0.4 ml
70839-4
71054-3
6
modeling (1, 2). Greater than 85% of the 1. Harrison, R. G. (2000) inNovations 11, 4–7.
2. Davis, G. D., Elisee, C., Newham, D. M., and
expressed protein was soluble in tests with each
Harrison, R. G. (1998) Biotechnol. Bioeng. 65, Rosetta-gami(DE3) 0.4 ml 71055-3
of four different NusA fusion proteins (1). The
382–388. Competent Cells 1 ml 71055-4
Rosetta-gami(DE3)pLysS
Competent Cells
0.4 ml
1 ml
71057-3
71057-4 7
inNovations No. 11
Appendix
www.novagen.com
8
10
11
Appendices
Indices

107
Novagen
Protein Expression
1 pET LIC Vector Kits Available pET LIC Vectors

High efficiency directional cloning into “gold standard” expression vectors Ek/LIC vectors
• pET-30
Ligation-independent cloning (LIC) was
developed for directional cloning of PCR prod- • pET-32
ucts without restriction enzyme digestion or liga- • pET-34
2 tion reactions (1, 2). The LIC method takes
advantage of the 3' → 5' exonuclease activity of
• pET-36
• pET-41
T4 DNA polymerase to create very specific 12 to
• pET-43.1
15 nucleotide single stranded overhangs in the
vector and the insert, so that the vast majority of Xa/LIC vectors
annealed products consist of the desired • pET-30
3 molecules. The annealed LIC vector and insert
• pET-32
are transformed into NovaBlue competent cells,
• pET-35
and covalent bonds are formed at the vector-
insert junctions within the cell to yield circular • pET-37
plasmid. Directional cloning of the insert is
Kit Components (20 rxn):
4 achieved with minimal non-recombinant back-
ground, and cloning is so efficient that virtually • 1 µg Ek/LIC or Xa/LIC Vector
all of the resulting colonies contain the desired • 8 µl Control Insert
• 25 U T4 DNA Polymerase, LIC-qualified
recombinant.
• 50 µl 10X T4 DNA Polymerase Buffer
PCR products with complementary over-
• 100 µl 100 mM DTT
hangs are created by building appropriate 5'
5 extensions into the primers. The purified PCR
products are treated with T4 DNA polymerase in Recombinant Enterokinase or Factor Xa.
• 50 µl
• 40 µl
• 1.5 ml
25 mM EDTA
25 mM dATP or dGTP
Nuclease-free Water
the presence of the appropriate dNTP to gener- The Ek/LIC and Xa/LIC vector kits provide
• 22 × 50 µl NovaBlue Singles™ Competent
ate the specific vector-compatible overhangs. the necessary reagents for creating single strand- Cells
Please see below for detailed primer design ed overhangs, annealing with the vector, and • 0.2 ml BL21(DE3) Competent Cells
information and the Appendix for diagrams of transforming competent E. coli cells. Each kit • 0.2 ml BL21(DE3)pLysS Competent Cells
6 the LIC strategies. provides enough reagents for 20 annealings and • 5 × 2 ml SOC Medium
transformations. A Control Insert is included to • 10 µl Test Plasmid
Two Different Protease Cleavage Sites
verify performance.
Two families of pET ligation-independent
cloning vectors are available: enterokinase 1. Aslanidis, C. and de Jong, P. J. (1990) Nucleic Acids
7 (Ek/LIC) and Factor Xa (Xa/LIC). With both

types of vector, the LIC site is designed to 2.
Res. 18, 6069–6074.
Haun, R. S., Servanti, I. M., and Moss, J. (1992)
enable removal of all vector-encoded sequences BioTechniques 13, 515–518.
from the target protein by digestion with either continued on next page
8
Primer design for expression of inserts in Ek/LIC and Xa/LIC Vectors
Ek/LIC for enterokinase cleavage Xa/LIC for Factor Xa cleavage
9 The Ek/LIC site in Ek/LIC vectors has a 13-base single stranded over-
hang on the left side and a 14-base single stranded overhang on the
The Xa/LIC site in Xa/LIC vectors has a 12-base single stranded over-
hang on the left side and a 15-base single stranded overhang on the
right side. The left side of the Ek/LIC site is designed to encode the right side. The left side of the Xa/LIC site is designed to encode the
recognition site for enterokinase (DDDDK↓). This feature enables recognition site for Factor Xa (IEGR↓). Like enterokinase, Factor Xa
removal of all of the vector encoded fusion sequences from expressed cleaves on the C-terminal side of its recognition site, thus a protein
proteins by cleavage with enterokinase. The following sequences must possessing any desired amino-terminus (except proline) can be gen-
10 be added to the 5' end of the target gene PCR primers to generate
vector-compatible overhangs:
erated by Factor Xa cleavage using these vectors. The following
sequences must be added to the 5' end of the target gene PCR
primers to generate vector-compatible overhangs:
Sense primer: 5'-GAC GAC GAC AAG ATX*
Antisense primer: 5'-GAG GAG AAG CCC GGT Sense primer: 5'-GGT ATT GAG GGT CGC
Antisense primer: 5'-AGA GGA GAG TTA GAG CC
The antisense primer may encode a stop codon or allow read-through
11 to the vector-encoded stop codon present after the C-terminal

His•Tag® sequence.
The sense primer encodes the Factor Xa recognition site and is in-
frame with the open reading frame (ORF) defined by the vector. Note
that the first three nucleotides of the insert-specific sequence must
* The first nucleotide of the insert-specific sequence must complete maintain this ORF and will encode the N-terminal amino acid of the
the codon ATX to give Ile (X = A, C or T) or Met (X = G). protein following cleavage with Factor Xa. The antisense primer may
Appendices
encode a stop codon or allow read-through to the vector-encoded stop

Indices
codon present after the C-terminal His•Tag sequence.

Novagen
Protein Expression
pET LIC Vector Kits continued Product Size Cat. No.

1
pET-30 Ek/LIC
The various pET Ek/LIC and Xa/LIC vectors pET-30 Xa/LIC
are shown below. Vector Kit 20 rxn 70073-3
pET-32 Ek/LIC
Vector Kit
pET-32 Xa/LIC
20 rxn 69076-3
2
pET-30 Ek/LIC enterokinase site Vector Kit 20 rxn 70072-3
Bpu1102 I
pET-30 Xa/LIC Factor Xa site
BamH I
EcoR V
Hind III
thrombin site
EcoR I
pET-34 Ek/LIC
Nde I
Nco I
Kpn I
Bgl II
Xho I
Sac I
Eag I
Not I
Sal I
T7lac His•Tag S•Tag LIC LIC His•Tag
pET-32 Ek/LIC enterokinase site

pET-35 Xa/LIC
Vector Kit 20 rxn 70115-3 3
Bpu1102 I
pET-32 Xa/LIC Factor Xa site
BamH I
EcoR V
thrombin site
Hind III
EcoR I pET-36 Ek/LIC
Msc I
Nco I
Kpn I
Bgl II
Xho I
Sac I
Eag I
Not I
Sal I
T7lac Trx•Tag His•Tag S•Tag LIC LIC His•Tag
pET-37 Xa/LIC
Bpu1102 I
pET-35 Xa/LIC thrombin site Factor Xa site
4
BamH I
EcoR V
Hind III
pET-41 Ek/LIC
EcoR I
SexA I
Mun I
Sac II
Nco I
Kpn I
Bgl II
Xho I
Avr II
Sca I
Sac I
Eag I
Not I
Sal I
T7lac CBDclos•Tag S•Tag LIC LIC His•Tag Vector Kit 20 rxn 71071-3
pET-43.1 Ek/LIC
Bpu1102 I
pET-37 Xa/LIC thrombin site Factor Xa site

BamH I
EcoR V
Hind III
EcoR I
SexA I
Mun I
Nde I
Nco I
Kpn I
Bgl II
Xho I
Avr II
Sca I
Sac I
Eag I
Not I
Available separately:
T7lac signal CBDcenA•Tag S•Tag LIC LIC His•Tag
Product Size Cat. No. 5
pET-41 Ek/LIC
Bpu1102 I
Sse8387 I
thrombin site enterokinase site (linearized vector)

BamH I
EcoR V
Hind III
EcoR I
BsrG I
PinA I
Mun I
Sac II
Nde I
Nco I
Kpn I
Spe I
Bgl II
Xho I
Avr II
Sac I
Eag I
Asc I
Not I
Stu I
Pst I

Sal I
T7lac GST•Tag His•Tag S•Tag LIC LIC His•Tag

(linearized vector)
pET-43.1 Ek/LIC thrombin site pET-32 Ek/LIC Vector 1 µg 69023-3

6
Sse8387 I
enterokinase site (linearized vector)

BamH I
Hind III
EcoR I
BsrG I
Sma I
Sac II
Nde I
Kpn I
Spe I
Xho I
Avr II
Pml I
Sac I
Eag I
Pac I
Asc I
Not I
Pst I
Sal I
T7lac Nus•Tag His•Tag S•Tag LIC LIC HSV•Tag His•Tag pET-32 Xa/LIC Vector 1 µg 70023-3
(linearized vector)
f1 o pET-34 Ek/LIC Vector 1 µg 70100-3

rig (linearized vector)
in
Kan Ap
7
l a cl
(linearized vector)
pET-30 Ek/LIC pET-32 Ek/LIC
pET-30 Xa/LIC pET-32 Xa/LIC pET-36 Ek/LIC Vector 1 µg 70134-3
pET-34 Ek/LIC pET-43.1 Ek/LIC* (linearized vector)
Kan
pET-35 Xa/LIC
Ap
pET-36 Ek/LIC pET-37 Xa/LIC Vector 1 µg 70136-3

pET-37 Xa/LIC (linearized vector)
pET-41 Ek/LIC
o ri * Ap gene is in opposite orientation in pET-43.1 Ek/LIC
pET-41 Ek/LIC Vector
(linearized vector)
1 µg 71069-3
8
pET-43.1 Ek/LIC Vector 1 µg 71070-3
(linearized vector)
T4 DNA Polymerase,
LIC-qualified 250 U 70099-3
NovaBlue Singles™
Competent Cells
11 rxn
22 rxn
70181-3
70181-4
9
Ek/LIC Vector Kit Protocol TB163
Xa/LIC Vector Kit Protocol
Ek/LIC Cloning Strategy
TB184
Appendix 10
Xa/LIC Cloning Strategy Appendix
inNovations No. 5
11
Appendices
Indices

109
Novagen
Protein Expression
1 pET Host Strain Glycerol Stocks Product Size Cat. No.
Pure stock cultures AD494 0.2 ml 69032-3

AD494(DE3) 0.2 ml 69020-3
All pET System host strains are available as BLR
AD494(DE3)pLysS 0.2 ml 69021-3
standard glycerol stocks. Many of these strains
BLR is a recA– derivative of BL21 that
are also available as pretested competent cells, AD494 Set 69043-3
improves plasmid monomer yields and may help (includes all 3 AD494 strains)
2 ready for transformation (see later in this chap-
ter). The designation (DE3) indicates that the
stabilize target plasmids containing repetitive B834 0.2 ml 69287-3
sequences or whose products may cause the loss
host is a lysogen of lDE3, and therefore carries B834(DE3) 0.2 ml 69288-3
of the DE3 prophage (5, 6).
a chromosomal copy of the T7 RNA polymerase B834(DE3)pLysS 0.2 ml 69289-3
gene under control of the lacUV5 promoter. HMS174
Such strains are suitable for production of pro- B834 Set 69044-3
3 tein from target genes cloned in pET vectors.
HMS174 strains provide the recA mutation in
a K-12 background. Like BLR, these strains may
(includes all 3 B834 strains)
BL21 0.2 ml 69386-3
pLysS and pLysE are designations given to hosts
stabilize certain target genes whose products
carrying pET-compatible plasmids that encode BL21(DE3) 0.2 ml 69387-3
may cause the loss of the DE3 prophage.
T7 lysozyme, which is a natural inhibitor of T7 BL21(DE3)pLysS 0.2 ml 69388-3
RNA polymerase. Cells containing pLysS pro- NovaBlue
BL21(DE3)pLysE 0.2 ml 69389-3
4 duce a small amount of T7 lysozyme, while
pLysE hosts produce much greater amounts.
NovaBlue is a K-12 strain ideally suited as an
initial cloning host due to its high transformation
BL21(DE3)pLacI 0.2 ml 70615-3
These strains are used to suppress basal expres- BL21 Set 69443-3
efficiency, blue/white screening capability (with (includes first 4 BL21 strains)
sion of T7 RNA polymerase prior to induction
appropriate plasmids) and recA endA mutations,
and thus stabilize pET recombinants encoding BL21trxB(DE3) 0.2 ml 70506-3
which result in high yields of excellent quality
target proteins that affect cell growth and viabili- BL21trxB(DE3)pLysS 0.2 ml 70507-3
5 ty. Hosts that carry the pLacI plasmid produce
extra lac repressor, which is required to sup-
plasmid DNA. The DE3 lysogen of NovaBlue is
potentially useful as a stringent host due to the
presence of the lacIq repressor encoded by the F
BLR 0.2 ml 69207-3
press basal expression from pETBlue™ and BLR(DE3) 0.2 ml 69208-3
episome. Blue/white screening is not possible
pTriEx™ vectors. The lDE3 Lysogenization Kit BLR(DE3)pLysS 0.2 ml 69209-3
with NovaBlue(DE3) due to the presence of lacZ
is also available for making new expression
a-peptide coding sequences in the lysogenic BLR Set 69057-3
hosts with other genetic backgrounds.
6 AD494
phage. (includes all 3 BLR strains)
HMS174 0.2 ml 69385-3
Origami™
AD494 strains are thioredoxin reductase HMS174(DE3) 0.2 ml 69391-3
Origami host strains are K-12 derivatives that
(trxB) mutants that enable disulfide bond forma- HMS174(DE3)pLysS 0.2 ml 69392-3
have mutations in both the thioredoxin reduc-
tion in the cytoplasm, providing the potential to
tase (trxB) and glutathione reductase (gor) HMS174(DE3)pLysE 0.2 ml 69393-3
7 produce properly folded, active proteins (1). The
trxB mutation is selectable on kanamycin; there-
genes, which together greatly enhance disulfide
bond formation in the cytoplasm. Studies have
HMS174 Set
(includes all 4 HMS174 strains)
69444-3
fore, these strains are recommended for use
shown that expression in Origami(DE3) yielded NovaBlue 0.2 ml 69009-3
with plasmids carrying the ampicillin resistance
10-fold more active protein than in another host
marker bla. Origami™ 0.2 ml 70616-3
even though overall expression levels were simi-
lar (7). Origami hosts are compatible with ampi- Origami(DE3) 0.2 ml 70617-3
8 B834
B834 is the parental strain for BL21 (2).
cillin-resistant plasmids and are ideal for use Origami(DE3)pLysS 0.2 ml 70618-3
with pET-32 vectors, since the thioredoxin
These protease deficient hosts are methionine Origami(DE3)pLacI 0.2 ml 70619-3
fusion tag further enhances the formation of
auxotrophs and allow high specific activity label- Origami Set 70668-3
disulfide bonds in the cytoplasm. The
ing of target proteins with 35S-methionine and (includes first 3 Origami strains)
Origami(DE3)pLacI host is compatible with
selenomethionine for crystallography (3). Origami B 0.2 ml 70829-3
9 BL21
expression from pETBlue™ and pTriEx™ vec-
tors. The trxB and gor mutations are selectable Origami B(DE3) 0.2 ml 70830-3
on kanamycin and tetracycline, respectively; Origami B(DE3)pLysS 0.2 ml 70832-3
BL21 is the most widely used host back-
therefore, these strains are recommended for
ground and has the advantage of being deficient Origami B(DE3)pLacI 0.2 ml 70831-3
use only with pET plasmids carrying the ampi-
in both lon (4) and ompT proteases.
cillin resistance marker bla. Origami B Set 70910-3
10 BL21trxB
Origami B
(includes first 3 Origami B strains)
glycerol stock listing continued on next page
BL21trxB strains possess the same thiore-
Origami B host strains carry the same
doxin reductase mutation (trxB) as the AD494
trxB/gor mutations as the original Origami
strains in the protease deficient BL21 back-
strains, except that they are derived from a
ground. Since trxB hosts facilitate cytoplasmic
lacZY mutant of BL21. Thus the Origami B
11 disulfide bond formation, their use may increase
the fraction of properly folded protein (1). The
strains combine the desirable characteristics of
BL21, Tuner™ and Origami hosts in one strain
trxB mutation is selectable on kanamycin; there-
background. The trxB and gor mutations are
fore, these strains are recommended for use only
selectable on kanamycin and tetracycline,
Appendices
with plasmids carrying the ampicillin resistance

respectively; therefore, these strains are recom-
Indices
marker bla.
mended for use only with pET plasmids carrying
the ampicillin resistance marker bla.

Novagen
Protein Expression
pET Host Strain Glycerol Stocks continued Product Size Cat. No.
1
Rosetta™ 0.2 ml 70949-3
Rosetta(DE3) 0.2 ml 70950-3
Rosetta™ AUA, CUA, CCC, GGA codons on a compatible
Rosetta(DE3)pLysS 0.2 ml 70951-3
chloramphenicol-resistant plasmid. The tRNA
Rosetta host strains are Tuner™ derivatives
genes are driven by their native promoters. In Rosetta(DE3)pLacI 0.2 ml 70952-3
designed to enhance the expression of eukaryot-
ic proteins that contain codons rarely used in
Rosetta-gami(DE3)pLysS and Rosetta-
gami(DE3)pLacI, the rare tRNA genes are pre-
Rosetta Set
(includes first 3 Rosetta strains)
70986-3 2
E. coli. These strains supply tRNAs for AGG,
sent on the same plasmids that carry the T7 RosettaBlue™ 0.2 ml 71065-3
AGA, AUA, CUA, CCC, GGA codons on a com-
lysozyme and lac repressor genes, respectively. RosettaBlue(DE3) 0.2 ml 71066-3
patible chloramphenicol-resistant plasmid. Thus
The trxB and gor mutations are selectable on
the Rosetta strains provide for “universal” trans- RosettaBlue(DE3)pLysS 0.2 ml 71068-3
kanamycin and tetracycline, respectively; there-
lation which is otherwise limited by the codon
usage of E. coli. The tRNA genes are driven by
fore, these strains are recommended for use only RosettaBlue(DE3)pLacI 0.2 ml 71067-3 3
with pET plasmids carrying the ampicillin resis- RosettaBlue Set 71081-3
their native promoters. In Rosetta(DE3)pLysS
tance marker bla. (includes first 3 RosettaBlue strains)
and Rosetta(DE3)pLacI, the rare tRNA genes are
present on the same plasmids that carry the T7 Rosetta-gami™ 0.2 ml 71061-3
Tuner™
lysozyme and lac repressor genes, respectively. Rosetta-gami(DE3) 0.2 ml 71062-3
RosettaBlue™
Tuner™ strains are lacZY deletion mutants
of BL21 and enable adjustable levels of protein Rosetta-gami(DE3)pLysS 0.2 ml 71064-3 4
expression throughout all cells in a culture. The Rosetta-gami(DE3)pLacI 0.2 ml 71063-3
RosettaBlue host strains are NovaBlue
lac permease (lacY) mutation allows uniform
derivatives that combine high transformation Rosetta-gami Set 71082-3
entry of IPTG into all cells in the population, (includes first 3 Rosetta-gami strains)
efficiency and recA endA lacIq mutations with
which produces a concentration-dependent, Tuner™ 0.2 ml 70611-3
enhanced expression of eukaryotic proteins that
contain codons rarely used in E. coli. These
strains supply tRNAs for AGG, AGA, AUA, CUA,
homogeneous level of induction. By adjusting
the concentration of IPTG, expression can be Tuner(DE3) 0.2 ml 70612-3 5
regulated from very low level expression up to Tuner(DE3)pLysS 0.2 ml 70613-3
CCC, GGA codons on a compatible chloram-
the robust, fully induced expression levels com- Tuner(DE3)pLacI 0.2 ml 70614-3
phenicol-resistant plasmid. The tRNA genes are
monly associated with pET vectors. Lower level
driven by their native promoters. Tuner Set 70667-3
expression may enhance the solubility and activ-
In RosettaBlue(DE3)pLysS and
RosettaBlue(DE3)pLacI, the rare tRNA genes are
ity of difficult target proteins. The
(includes first 3 Tuner strains)
6
Tuner(DE3)pLacI strain is compatible with
present on the same plasmids that carry the T7 Additional Information Available
expression from pETBlue™ and pTriEx™ vec-
lysozyme and lac repressor genes, respectively.
tors. pET System Manual TB055
Blue/white screening is not possible with Host Strain Genotypes Appendix
RosettaBlue(DE3) strains due to the presence of 1. Derman, A. I., Prinz, W. A., Belin, D. and Beckwith, Patent and Licensing Information p. 278
lacZ a-peptide coding sequences in the DE3 lyso-
genic phage. 2.
J. (1993) Science 262, 1744–1747.
Wood, W. B. (1966) J. Mol. Biol. 16, 118–133.
7
3. Leahy, D. J., Hendrickson, W. A., Aukhil, I., and
Rosetta-gami™ Erickson, H. P. (1992) Science 258, 987–991.
Rosetta-gami™ host strains are Origami™ 4. Phillips, T. A., Van Bogelen, R. A., and Neidhardt, F.
C. (1984) J. Bacteriol. 159, 283–287.
derivatives that combine the enhanced disulfide
bond formation resulting from trxB/gor muta-
5.
6.
A. Roca (U. of Wisconsin), personal communication.
Studier, F. W. (1991) J. Mol. Biol. 219, 37–44.
8
tions with enhanced expression of eukaryotic
proteins that contain codons rarely used in E. 7. Prinz, W. A., Aslund, F., Holmgren, A., and
Beckwith, J. (1997) J. Biol. Chem. 272,
coli. These strains supply tRNAs for AGG, AGA, 15661–15667.
100 mM IPTG Solution Product Size Cat. No.
Convenient formulation for inducing protein expression 100 mM IPTG Solution 15 ml 70527-3
(10 × 1.5 ml)
10
The lac operon inducer IPTG (isopropyl-b-D- preparation is dioxane-free and supplied as a
galactopyranoside) is recommended for protein sterile concentrate (100 mM) in water, in conve-
expression with the pET System and other lac nient 1.5 ml aliquots. Store at –20°C.
promoter-derived expression systems. The
11
Appendices
Indices

111
Novagen
Protein Expression
1 pET Host Strain Competent Cells

Provided in 0.2 ml aliquots, ready for efficient transformation
pET Host Strain Competent Cells are avail- Product Size Cat. No.
Expression Strains: lDE3 Lysogens
able for convenient, efficient construction of for protein expression using pET vectors RosettaBlue™(DE3) 0.4 ml 71059-3
pET recombinants. The cells are grown and Competent Cells 1 ml 71059-4
2 made competent by an optimized procedure, fol-
lowed by verification of transformation efficien- AD494(DE3) 0.4 ml 69013-3
guaranteed efficiency 1 × 108 cfu/µg
RosettaBlue(DE3)pLysS 0.4 ml 71034-3
Competent Cells 1 ml 69013-4 Competent Cells 1 ml 71034-4
cy and strain identity (not intended for electro- guaranteed efficiency 2 × 106 cfu/µg 8
poration).
AD494(DE3)pLysS 0.4 ml 69014-3 Rosetta-gami™(DE3) 0.4 ml 71055-3
Features Competent Cells 1 ml 69014-4 Competent Cells 1 ml 71055-4
3 • Provided as frozen 0.2 ml aliquots; each vial
can be used for ten 20 µl transformations B834(DE3) 0.4 ml 69041-3
Rosetta-gami(DE3)pLysS 0.4 ml 71057-3
• Includes Test Plasmid, SOC Medium and guaranteed efficiency 2 × 106 cfu/µg
protocol B834(DE3)pLysS 0.4 ml 69042-3 Tuner™(DE3) 0.4 ml 70623-3
• Reproducible high efficiencies Competent Cells 1 ml 69042-4 Competent Cells 1 ml 70623-4
4 • Selection of expression strains (lDE3
lysogens) and isogenic control strains
BL21(DE3)
Competent Cells
0.4 ml
1 ml
69450-3
69450-4
Tuner(DE3)pLysS 0.4 ml 70624-3
guaranteed efficiency 2 × 106 cfu/µg Competent Cells 1 ml 70624-4
(nonlysogens) 6
BL21(DE3)pLysS 0.4 ml 69451-3
Competent Cells 1 ml 69451-4 Isogenic Strains (non-lDE3 Lysogens)
guaranteed efficiency 2 × 106 cfu/µg for initial pET cloning (NovaBlue), isogenic controls and
5 BL21trxB(DE3)
Competent Cells
0.4 ml
1 ml
70508-3
70508-4
protein expression using non-T7 vectors

AD494 0.4 ml 69033-3
BL21trxB(DE3)pLysS 0.4 ml 70509-3 Competent Cells 1 ml 69033-4
Competent Cells 1 ml 70509-4 guaranteed efficiency 2 × 106 cfu/µg
6
BL21 0.4 ml 69449-3
6 BLR(DE3)
Competent Cells
0.4 ml
1 ml
69053-3
69053-4
Competent Cells 1 ml
69449-4
BLR 0.4 ml 69052-3

BLR(DE3)pLysS 0.4 ml 69956-3 Competent Cells 1 ml 69052-4
HMS174 0.4 ml 69452-3
HMS174(DE3) 0.4 ml 69453-3
7 Competent Cells 1 ml
69453-4
Competent Cells 1 ml
69452-4
NovaBlue 0.4 ml 69825-3

HMS174(DE3)pLysS 0.4 ml 69454-3 Competent Cells 1 ml 69825-4
Competent Cells 1 ml 69454-4 8
Origami™ 0.4 ml 70626-3
NovaBlue(DE3) 0.4 ml 69284-3 Competent Cells 1 ml 70626-4
8 Competent Cells 1 ml
69284-4 6
Origami B 0.4 ml 70836-3
Origami™(DE3) 0.4 ml 70627-3 Competent Cells 1 ml 70836-4
Rosetta™ 0.2 ml 70953-3
Origami(DE3)pLysS 0.4 ml 70628-3 Competent Cells 1 ml 70953-4
9 guaranteed efficiency 2 × 106 cfu/µg
RosettaBlue™ 0.4 ml 71058-3
Origami B(DE3) 0.4 ml 70837-3 Competent Cells 1 ml 71058-4
Rosetta-gami™ 0.4 ml 71054-3
Origami B(DE3)pLysS 0.4 ml 70839-3 Competent Cells 1 ml 71054-4
10 guaranteed efficiency 2 × 106 cfu/µg
Tuner™ 0.4 ml 70622-3
Rosetta™(DE3) 0.2 ml 70954-3 Competent Cells 1 ml 70622-4
Rosetta(DE3)pLysS 0.4ml 70956-3
Competent Cells 1 ml 70956-4 Additional Information Available
11 6
Competent Cells Protocol
pET System Manual
TB009
TB055
Host Strain Genotypes Appendix
Appendices
Indices

Novagen
Protein Expression
pET Competent Cell Sets 1

Sets of pET host strain competent cells for optimizing pET vector/host strain combinations
pET Competent Cell Sets consist of popular Product Cat. No. Product Cat. No.
strains that are often used side-by-side in opti-
(DE3) Competent Cell Set 71032-3 Origami™ Competent Cell Set 70670-3
mization experiments.
AD494(DE3), BL21(DE3), BL21trxB(DE3), Tuner(DE3),
BLR(DE3), HMS174(DE3), NovaBlue(DE3), Origami(DE3),
Origami B(DE3), Rosetta(DE3);
Origami, Origami(DE3), Origami(DE3)pLysS;
2 × 0.2 ml each, SOC and Test Plasmid 2
0.2 ml each, SOC and Test Plasmid Origami B Competent Cell Set 70911-3
Origami B, Origami B(DE3), Origami B(DE3)pLysS;
(DE3)pLysS Competent Cell Set 71033-3 2 × 0.2 ml each, SOC and Test Plasmid
AD494(DE3)pLysS, BL21(DE3)pLysS, BL21trxB(DE3)pLysS,
Tuner(DE3)pLysS, BLR(DE3)pLysS, HMS174(DE3)pLysS, Rosetta™ Competent Cell Set 70987-3
Origami(DE3)pLysS, Origami B(DE3)pLysS,
Rosetta(DE3)pLysS;
Rosetta, Rosetta(DE3), Rosetta(DE3)pLysS;
2 × 0.2 ml each, SOC and Test Plasmid
3
0.2 ml each, SOC and Test Plasmid
RosettaBlue™
AD494 Competent Cell Set 70231-3 Competent Cell Set 71079-3
AD494, AD494(DE3), AD494(DE3)pLysS; RosettaBlue, RosettaBlue(DE3), RosettaBlue(DE3)pLysS;
2 × 0.2 ml each, SOC and Test Plasmid 2 × 0.2 ml each, SOC and Test Plasmid
BL21 Competent Cell Set 70232-3 Rosetta-gami™ 4
BL21, BL21(DE3), BL21(DE3)pLysS; Competent Cell Set 71080-3
2 × 0.2 ml each, SOC and Test Plasmid Rosetta-gami, Rosetta-gami(DE3),
Rosetta-gami(DE3)pLysS;
BLR Competent Cell Set 70233-3 2 × 0.2 ml each, SOC and Test Plasmid
BLR, BLR(DE3), BLR(DE3)pLysS;
HMS174 Competent Cell Set 70234-3
Tuner™ Competent Cell Set
Tuner, Tuner(DE3), Tuner(DE3)pLysS;
70726-3
5
HMS174, HMS174(DE3), HMS174(DE3)pLysS;
Competent Cells Protocol TB009
pET System Manual
Host Strain Genotypes
TB055
Appendix
6
pETBlue™ and pTriEx™ Host Strain Competent Cells

Hosts specifically designed for T7-driven protein expression with pETBlue and pTriEx vectors 7
Hosts that carry the pLacI plasmid produce Expression Strains Isogenic Strains (non-lDE3 Lysogens)
extra lac repressor, which is required to sup- for protein expression using pETBlue and pTriEx vectors for initial pET cloning (NovaBlue) and as isogenic controls
press basal expression from pETBlue™ and
Product Size Cat. No. Product Size Cat. No.
pTriEx™ vectors.
Features
Origami™(DE3)pLacI
Competent Cells
0.4 ml
1 ml
70629-3
70629-4
NovaBlue
Competent Cells
0.4 ml
1 ml
69825-3
69825-4
8
• Choice of popular strains for use with guaranteed efficiency 2 × 106 cfu/µg guaranteed efficiency 1 × 108 cfu/µg
pETBlue and pTriEx vectors Origami B(DE3)pLacI 0.4 ml 70838-3 Origami 0.4 ml 70626-3
• Provided as frozen 0.2 ml aliquots; each vial
guaranteed efficiency 2 × 106 cfu/µg guaranteed efficiency 2 × 106 cfu/µg
can be used for ten 20 µl transformations
• Includes Test Plasmid, SOC Medium and
Rosetta™(DE3)pLacI
Competent Cells
6
0.4 ml
1 ml
70920-3
70920-4
Origami B
Competent Cells
0.4 ml
1 ml
70836-3
70836-4 9
protocol guaranteed efficiency 2 × 106 cfu/µg
• Reproducible high efficiencies Rosetta-gami™(DE3)pLacI Rosetta 0.2 ml 70953-3

0.4 ml 71056-3 Competent Cells 1 ml 70953-4
• Selection of expression strains (lDE3 Competent Cells 1 ml 71056-4 6
lysogens) and isogenic control strains guaranteed efficiency 2 × 106 cfu/µg
RosettaBlue 0.4 ml 71058-3
(nonlysogens) RosettaBlue™(DE3)pLacI
0.4 ml 71060-3
Competent Cells
8
1 ml
71058-4 10
• Note: these strains are not recommended for
use with pET vectors (pETBlue and pTriEx guaranteed efficiency 1 × 108 cfu/µg Rosetta-gami 0.4 ml 71054-3
vectors have a different plasmid backbone Competent Cells 1 ml 71054-4
Tuner™(DE3)pLacI 0.4 ml 70625-3 6
than the pET vectors) Competent Cells 1 ml 70625-4
6 Tuner 0.4 ml 70622-3
Competent Cells
6
1 ml
70622-4 11
Appendices

Indices
pETBlue System Protocol TB249

TriEx System Manual TB250

113
Novagen
Protein Expression
1 Singles™ Competent Cells

High efficiency transformation in a one-tube format
Singles™ Competent Cells are ready-to-use NovaBlue

Expression Strains: lDE3 Lysogens
single aliquots designed for high efficiency, for protein expression using pET vectors for initial cloning into pET, pETBlue and pTriEx vectors
ultra-convenient transformation.
2 Features
Product
BL21(DE3) Singles™
Size
11 rxn
Cat. No.
70235-3 NovaBlue Singles 11 rxn 70181-3
• Provided as frozen single-use 50 µl aliquots in Competent Cells 22 rxn 70235-4 Competent Cells 22 rxn 70181-4
11 reaction or 22 reaction kits guaranteed efficiency > 2 × 106 cfu/µg guaranteed efficiency > 1.5 × 108 cfu/µg
• Includes Test Plasmid, SOC Medium and BL21(DE3)pLysS Singles 11 rxn 70236-3
protocol Competent Cells 22 rxn 70236-4 Additional Information Available
6
guaranteed efficiency > 2 × 10 cfu/µg
3 • Reproducible high efficiencies
Origami™(DE3) Singles 11 rxn 70630-3
Competent Cells Protocol
pET System Manual
TB009
TB055
• Nothing to add except DNA Competent Cells 22 rxn 70630-4 Host Strain Genotypes Appendix
• No need to subaliquot; perform Patent and Licensing Information p. 278
transformation right in the supplied tube Origami(DE3)pLysS
Singles 11 rxn 70631-3
containing the cells Competent Cells 22 rxn 70631-4
4 • Selection of pET expression strains (λDE3
lysogens) and NovaBlue for initial cloning
Rosetta™(DE3) Singles 11 rxn 71099-3
into pET, pETBlue™ and pTriEx™ vectors Competent Cells 22 rxn 71099-4
Rosetta(DE3)pLysS 11 rxn 71100-3
Singles Competent Cells 22 rxn 71100-4
5 guaranteed efficiency > 2 × 106 cfu/µg
6
HT96™ Competent Cells
Pre-dispensed in a 96-well format for high throughput cloning and expression
HT96™ Competent Cells are designed for BL21(DE3) NovaBlue

7 convenient, high efficiency transformation in a for protein expression using pET vectors for initial cloning into pET, pETBlue and pTriEx vectors
96-well format.
Product Size Cat. No. Product Size Cat. No.
Features
HT96™ BL21(DE3) 1 plate 71012-3 HT96™ NovaBlue 1 plate 71011-3
• Perform high efficiency transformation Competent Cells 4 plates 71012-4 Competent Cells 4 plates 71011-4
directly in the plate 20 plates 71012-5 20 plates 71011-5
8 • Provided as frozen 20 µl aliquots in
Components:
Components:
96-well polypropylene plates
• 1 or 4 or 20 plates HT96 Competent Cells • 1 or 4 or 20 plates HT96 Competent Cells
• 0.2 ml well configuration compatible with • 1 or 4 or 20 × 14 ml SOC Medium • 1 or 4 or 20 × 14 ml SOC Medium
HT96 Isothermal Block and many thermal • 1 or 2 or 10 × 10 µl Test Plasmid • 1 or 2 or 10 × 10 µl Test Plasmid
cyclers • 1 or 4 or 20 pkg/12 8-Cap Strips • 1 or 4 or 20 pkg/12 8-Cap Strips
9 • Wells individually sealed; seal may be peeled
off or pierced
• 1 or 4 or 20 Reagent Reservoirs • 1 or 4 or 20 Reagent Reservoirs
• Cap strips provided for resealing Product Cat. No.
• Groups of 24 wells may be cut from the plate HT96 Isothermal Block 71031-3
• Includes SOC Medium and reservoir
10 Additional Information Available
11
Appendices
Indices

Novagen
Protein Expression
Bacteriophage CE6 Product Size Cat. No.

1
λ phage for delivery of T7 RNA polymerase Bacteriophage CE6 0.2 ml 69390-3
10 ml 69390-4
Bacteriophage CE6 is a recombinant lambda that are not DE3 lysogens. The phage has lcI857 Components:
phage containing the T7 RNA polymerase gene immunity and the Sam7 mutation, which • 0.2 or 10 ml CE6 Lysate
(1). The polymerase gene is cloned into the int requires supF hosts for lytic growth (neither • 0.2 ml LE392 glycerol stock
gene, such that it is transcribed from the pL and
pI promoters of the phage during infection. lCE6
lytic growth nor different immunity are required
for T7 RNA polymerase synthesis). It is provided
• 0.2 ml ED8739 glycerol stock
2
can be used to provide a source of T7 RNA poly- as a high titer lysate and includes host strains Additional Information Available
merase to susceptible host cells carrying pET, LE392 (rK12– mK12+) and ED8739 (rK12– mK12–) as
Bacteriophage CE6 Protocol TB007
pETBlue™, or pTriEx™ recombinant plasmids. glycerol stocks. The 10 ml size is enough to pET System Manual TB055
In general, it is used when a target gene is so induce up to 100 ml culture. pETBlue System Protocol TB249
toxic that the plasmid cannot be maintained in
1. Studier, F. W. and Moffatt, B. A. (1986) J. Mol. Biol.
TriEx System Manual
TB250
p. 278
3
DE3 lysogenic hosts, or with alternative hosts 189, 113–130.
6
λDE3 Lysogenization Kit Product Size Cat. No.
Construction of bacterial strains that carry inducible T7 RNA polymerase lDE3 Lysogenization Kit 10 rxn 69734-3
lDE3 Lysogenization Kit
The lDE3 Lysogenization Kit is designed for on cells that lack T7 RNA polymerase, but plus pLysS and pLysE 10 rxn 69725-3
site-specific integration of lDE3 prophage into makes normal plaques on lDE3 lysogens in the Components: 7
an E. coli host cell chromosome, such that the presence of IPTG. An optional configuration • 50 µl lDE3 Phage Lysate
lysogenized host can be used to express target includes plasmids pLysS and pLysE (1 µg each), • 50 µl Helper Phage Lysate
genes cloned in pET vectors. lDE3 is a recombi- which can be transformed into the host for addi- • 50 µl Selection Phage Lysate
nant phage carrying the cloned gene for T7 RNA tional control over basal expression levels. • 50 µl T7 Tester Phage Lysate
polymerase under lacUV5 control. Lysogens are
prepared by co-infection with the three provided
The T7 RNA Polymerase Monoclonal
Antibody can be used to verify the presence of
• 200 µl HMS174(DE3) glycerol stock
8
phages, and can be verified by plating the T7 T7 RNA polymerase in the host strain by
Tester Phage (a T7 RNA polymerase deletion Western blot analysis.
mutant). This phage is unable to make a plaque pLysS DNA 1 µg 69659-3
pLysE DNA 1 µg 69658-3
T7 RNA Polymerase
Monoclonal Antibody
50 µg
250 µg
70566-3
70566-4
9
λDE3 Lysogenization Kit Protocol TB031
pLysS and pLysE map
TB107
p. 278
10
11
Appendices
Indices

115
Novagen
Protein Expression
1 Insect Cell Expression Overview

Baculovirus, transient and stable expression systems
Novagen offers three basic options for pro- tageous to allow more complete protein modifi- selectable marker such as neomycin resistance.
tein expression in insect cells: the lytic bac- cation (such as glycoprotein processing) and is Stable cell lines are then established from indi-
ulovirus system, continuous expression from sta- obtained by using alternative baculovirus pro- vidual drug-resistant colonies. The majority of
2 bly transfected cells, and vectors that also
enable transient expression in transfected cells.
moters (e.g., gp64 and ie1 promoters). Novagen’s
BacVector® System offers a complete and unique
resistant cells will express the protein of interest
at various levels. Such cell lines maintain stable
line of transfer plasmids, linearized baculovirus expression for many passages (greater than 50),
BacVector® Baculovirus Expression System
DNA, insect cells and transfection reagents for enabling long term culture for the accumulation
The highest levels of foreign protein expres- maximum utility in baculovirus expression. and study of the expressed protein.
sion in insect cells are typically obtained with
3 the baculovirus expression vector system
Continuous Expression in
Stably Transfected Cell Lines
Transient Expression
(BEVS). To express a target protein, the insect Transient expression represents the most
cells are infected with a recombinant bac- Continuous protein expression using stably rapid method and is obtained simply by transfec-
ulovirus bearing the gene of interest. The infect- transfected insect cell lines is particularly useful tion of insect cell expression vectors containing
ed cells undergo a burst of protein expression, for the study of glycoproteins, secreted proteins appropriate promoters (e.g., gp64 and ie1 pro-
4 after which the cells die and may lyse. High level
expression is obtained by the use of very late
and membrane proteins such as receptors. In
this system the gene of interest is cloned into a
moters) in the absence of selection. With this
method it is important to optimize the transfec-
baculovirus promoters (e.g., polh and p10 pro- vector that utilizes a promoter recognized by the tion conditions to obtain high efficiency trans-
moters), which are only active in infected insect insect cell transcription machinery (e.g., bac- fection. Expression will typically peak between
cells during the final stages of the infection ulovirus ie1 or gp64 promoters). The expression 24 and 48 hours after transfection.
cycle. Expression at earlier times can be advan- vector is cotransfected into insect cells with a
5
Baculovirus Transfer Plasmids
Common features: • High-copy plasmid origin of replication • f1 origin of replication (in pBAC™ vectors)
• polh locus of integration • Ampicillin resistance marker • ss = signal sequence
6 • Compatible with BacVector-1000, -2000, and • pBACgus versions express GUS for color • SP = signal peptidase
-3000 Triple Cut DNA identification of baculovirus recombinants
Period of Fusion Tags Protease

Vector Promoter Expression N-terminal C-terminal Cleavage Sites Special Features/Applications
7 pBAC-1, pBACgus-1
pBAC-2cp, pBACgus-2cp
polh
polh
24–72 h
24–72 h
none
His•Tag/S•Tag™
His•Tag
His•Tag
none
Tb/Ek
“Classic” transfer plasmids for high-level expression; choice of
fusion tags and cloning sites. Three plasmids available as LIC
pBAC-7, pBACgus-7 polh 24–72 h CBDclos•Tag™/S•Tag His•Tag Tb/Ek vectors.
pBAC-8, pBACgus-8 polh 24–72 h CBDclos•Tag/S•Tag His•Tag Tb/Ek
pBAC-9, pBACgus-9 polh 24–72 h T7•Tag® CBDclos•Tag/His•Tag Tb
pBAC-3, pBACgus-3 polh 24–72 h ss/His•Tag/S•Tag His•Tag SP/Tb/Ek Produce signal sequence fusions to facilitate secretion of target
8 pBAC-10, pBACgus-10
pBAC4x-1, pBACgus4x-1
polh
polh/p10
24–72 h
24–72 h
ss/CBDcenD•Tag/S•Tag
none
His•Tag
none
SP/Tb/Ek
none
proteins to the medium. High level late production.
Four promoters/cloning sites for high expression of multiple target

genes in same cell.
pBACsurf-1 polh 24–72 h ss (signal sequence) gp64 none For display of target proteins as gp64 fusions on the virion surface.
Baculovirus Transfer Plasmids also Useful for Stable and Transient Expression
9 pBAC-5, pBACgus-5
pBAC-6, pBACgus-6
gp64
gp64
4–48 h
4–48 h
His•Tag/S•Tag
ss/His•Tag/S•Tag
His•Tag
His•Tag
Tb/Ek
SP/Tb/Ek
Mid level expression over long period post-infection for more
complete processing, with options for secretion.
pBAC-11, pBACgus-11 gp64 4–48 h ss/CBDcenD•Tag/S•Tag His•Tag SP/Tb/Ek
pAcP(+)IE1-1 ie1 4–16 h none none none Very early low level expression for complete processing. Vectors
pAcP(+)IE1-2 ie1 4–16 h none none none differ in providing vector-encoded ATG and selection of cloning
pAcP(+)IE1-3 ie1 4–16 h none none none sites.
10 pAcP(+)IE1-4
pAcP(–)IE1-5
ie1
ie1
4–16 h
4–16 h
none
none
none
none
none
none Very early low level expression, plus compatibility with larval
pAcP(–)IE1-6 ie1 4–16 h none none none infection.
pIE1 Vectors for Stable and Transient Expression

11 Common features:
Vector Provides ATG MCS Orientation Special Features/Applications
• Ampicillin resistance marker
pIE1-1 yes BsaB I–BamH I Low level continuous expression in
• High-copy plasmid origin of replication pIE1-2 yes BamH I–BsaB I transfected insect cells.
Appendices
• hr5 enhancer element pIE1-3 no BsaB I–BamH I

Indices
• Cotransfection with pIE1-neo enables pIE1-4 no BamH I–BsaB I

selection of recombinants with G418 pIE1-neo NA NA Selectable marker for cotransfection

Novagen
Protein Expression
BacVector® System 1
Complete baculovirus system for versatile protein expression
The baculovirus Autographa californica kits and conjugates for rapid, sensitive assay
ers
nuclear polyhedrosis virus (AcNPV) has become and affinity purification of fusion proteins ark
ers rn M
ark es te
a popular vehicle for cloning and expressing even in the absence of antibodies or other in™M in W in
te ote ote
recombinant proteins in insect cells (1–3).
Novagen offers the BacVector® System, a com-
target protein-specific reagents (see Figure
1); and the Ni-NTA His•Bind® Resins and kDa
Per
fec
t Pro
Perfect Pr
total cl
e l pr
2
plete set of reagents and kits for construction of Buffer Kit for economical purification based 150 —
recombinant baculoviruses and expression of on metal chelation chromatography. 100 —
proteins using advanced vectors. The BacVector 75 — — GUS
System provides a number of key improvements Construction of Recombinant Baculoviruses
over other baculovirus systems, including: A general scheme for constructing bac-
50 —
35 —
3
• BacVector Transfection Kits, including ulovirus recombinants is shown in Figure 2. In
25 —
BacVector-1000, BacVector-2000, or the first step, the target gene (shown as a PCR-
BacVector-3000 Triple Cut Virus DNA derived DNA) is cloned downstream of an
and Eufectin™ Transfection Reagent; AcNPV promoter in a suitable plasmid transfer 15 —
BacVector-2000 has been deleted for several
non-essential genes that compete with target
vector (e.g., pBAC). The transfer plasmid has
upstream and downstream segments of bac- Coomassie blue S•Tag™ LumiBlot™
4
protein production. BacVector-3000 has ulovirus DNA flanking the promoter and target
additional deletions of the v-cath and gene. Figure 1. Expression of b-glucuronidase (GUS) from a
chitinase genes. The v-cath protease deletion In the second step, the recombinant transfer BacVector-1000/pBAC-2cp Ek/LIC recombinant
stabilizes expressed proteins in crude lysates plasmid is co-transfected with linearized bac-
(4). ulovirus vector DNA (BacVector Triple Cut Virus
DNA) into insect cells. The flanking regions of
5
• pBAC™ and pBACgus Transfer Plasmids,
the transfer plasmid participate in homologous
and E. coli LIC Kits, for convenient cloning
recombination with the virus DNA sequences
and high level expression, including fusion
and introduce the target gene into the bac-
tags for detection and purification, optional target gene
ulovirus genome at the polyhedrin locus, repair-
gus marker gene for positive identification of
recombinants, and co-expression of multiple
ing the circular virus DNA and allowing viral +
pBAC LIC plasmid
6
replication to proceed. Typically, > 95% of the
genes. E. coli LIC (ligation-independent
plaques are recombinants. For additional verifi- gus
1629
cloning) kits contain prepared LIC transfer
cation, the pBACgus transfer plasmids contain a
plasmid, competent E. coli cells, buffers and pBAC E. coli LIC Kit
gus marker gene, which allows visual identifica-
controls.
• Choice of early, early/late, or very late
tion of recombinant plaques by their blue
appearance after staining with X-gluc.
pBAC transfer plasmid
7
gus
promoters in transfer plasmids. Following transfection and plaque purifica- target gene 1629
• Secretion vectors with signal peptide tion to remove parental virus, a high titer virus +
and early/late tandem promoter control, stock is prepared from the appropriate recombi- BacVector Triple Cut Virus DNA
for enhanced glycosylation and processing of nant. This stock is used to determine the optimal
secreted proteins. times for target protein expression (depending

29
8
• CBD•Tag™ sequences for rapid, on the promoter and the properties of the gene lacZ 16
economical protein purification based on the product). After these parameters are established,
cellulose binding domain, available for a large-scale culture is prepared and used for pro- BacVector Transfection Kit
cytoplasmic or secreted expression. tein production.

• Compatibility with many pTriEx™
Multisystem Vectors, which carry a very
1. Smith, G. E., Summers, M. D. and Fraser, M. J. gus
target gene 1629
9
(1983) Mol. Cell. Biol. 3, 2156–2165.
Recombinant virus DNA
late baculovirus promoter and recombina- 2. O’Reilly, D. R., Miller, L. K., and Luckow, V. A.
tion sequences for baculovirus construc- (1992) Baculovirus Expression Vectors: a Figure 2. Construction of baculovirus recombinants
Laboratory Manual, W. H. Freeman and Co., New with the BacVector System
tion, in addition to signals for expression
York.
•
in bacteria and mammalian cells.
FastPlax™ Titer Kit, for rapid colori-
3. King, L. A., and Possee, R. D. (1992) The
Baculovirus Expression System: A Laboratory
10
metric titering of baculovirus stocks. Manual, Chapman and Hall, UK.
• Compatibility with popular protein 4. Monsma, S. A., and Scott, M. D. (1997) inNovations
7, 16–19.
detection and purification reagents, such
as Novagen’s S•Tag™ System; a variety of
11
Appendices
Indices

117
Novagen
Protein Expression
1 BacVector® Transfection Kits Product Size Cat. No.
Convenient high-efficiency construction of recombinant baculoviruses BacVector®-1000

Transfection Kit 12 rxn 70059-3
BacVector® Transfection Kits are designed Features BacVector-2000
for efficient and reliable construction of bac- Transfection Kit 12 rxn 70030-3
• Greater than 95% recombinants
ulovirus recombinants starting with pBAC™, • BacVector-2000 DNA engineered to remove BacVector-3000
2 pTriEx™, pAcPIE1, or other compatible transfer
plasmid DNA. The BacVector Triple Cut Virus
nonessential genes
Transfection Kit
Components:
12 rxn 70077-3
• BacVector-3000 DNA engineered to remove

DNA has been digested with a restriction • 2 × 0.6 µg BacVector-1000, BacVector-2000
additional genes encoding proteolytic and or BacVector-3000 Triple Cut
enzyme that cuts within the essential gene
degradative enzymes Virus DNA
ORF1629, upstream of the lacZ gene and polh
• Presence of lacZ gene for easy visual • 2 µg Transfection Control Plasmid
promoter, and within the lacZ gene (1).
3 BacVector Triple Cut Virus DNA thus has an identification of recombinants • 3 vials
• 2×3g
Sf9 Insect Cells
BacPlaque™ Agarose
extremely low non-recombinant background, • Low cost per transfection
• 2 × 30 µl Eufectin™ Transfection Reagent
and typically produces nearly 100% recombinant • Compatible with pBAC and pTriEx plasmids • 2 × 500 µl X-Gluc Solution
plaques. BacVector Transfection Kits also con- and many other transfer vectors • 1.5 ml Nuclease-free Water
tain the Eufectin™ Transfection Reagent, Sf9
1. Kitts, P. A. and Possee, R. D. (1993) BioTechniques
4 Insect Cells, BacPlaque™ Agarose, and a
Transfection Control Plasmid, all of which have
14, 810–817.
Product
BacVector-1000
Size Cat. No.
been qualified for optimal transfection perfor- DNA Kit 12 rxn 70057-3
mance. BacVector-2000
BacVector DNA Kits contain the appropriate DNA Kit 12 rxn 70058-3
Triple Cut Virus DNA and matched Eufectin
5 Transfection Reagent.
BacVector-3000
DNA Kit 12 rxn 70078-3
Components:
1 2 3 4 M 5 6 7 8 9 • 2 × 0.6 µg BacVector Triple Cut Virus DNA
1. BacVector-1000, fresh • 2 × 30 µl Eufectin Transfection Reagent
kDa 2. BacVector-1000, 4 wks • 2 µg Transfection Control Plasmid
3. BacVector-2000, fresh
6 150 –
100 –
4.
M.
BacVector-2000, 4 wks
Perfect Protein™ Markers
75 – 5. BacVector-3000, fresh
6. BacVector-3000, 4 wks
Sf9 Insect Cells 3 vials 71104-3
50 – 7. BacVector-3000, fresh Eufectin Transfection
35 – 8. BacVector-3000, 4 wks Reagent 30 µl 70035-3
7 25 –
9. Sf9 cells, 4 wks
BacPlaque™ Agarose 30 g 70034-3
15 – Additional Information Available

BacVector Transfection Kits Protocol TB216
BacPlaque Agarose Protocol TB156
8 Effect of v-cath protease gene deletion in BacVector-3000 on infected Sf9 cell lysates
Sf9 cells were infected with BacVector-1000, BacVector-2000, or two BacVector-3000 recombinant viruses expressing various recombinant fusion
Sf9 Insect Cells Protocol
inNovations
TB329
Nos. 4a, 7
proteins. At 72 hours post infection, soluble crude cell lysates were prepared, and samples immediately taken (fresh). The remainder of the solu-
ble lysates were stored at 4°C for 4 weeks, and then samples were taken (4 wks). Three duplicate SDS-PAGE gels (4–20% gradient) were run;
samples contained equivalent amounts of protein.
10
11
Appendices
Indices

Novagen
Protein Expression
Baculovirus Transfer Plasmids Product Size Cat. No.

1
For convenient cloning and expression pAcP(+)IE1-1 DNA 10 µg 69092-3
pAcP(+)IE1-2 DNA 10 µg 69093-3
Novagen’s baculovirus transfer plasmids are ularly those involved in glycosylation and secre-
pAcP(+)IE1-3 DNA 10 µg 69094-3
designed for convenient cloning and expression tion (1). Thus, higher levels of biologically active
of target genes from baculovirus vectors. protein may be expressed from these vectors pAcP(+)IE1-4 DNA 10 µg 69095-3
Optional features include fusion tags for detec-
tion and purification, gus marker gene for posi-
than from classical polh-based vectors. The
pAcPIE1 series of transfer plasmids are based on
pAcP(–)IE1-5 DNA 10 µg 70170-3 2
pAcP(–)IE1-6 DNA 10 µg 69097-3
tive identification of recombinants, and co- the ie1 immediate early promoter combined with
expression of multiple genes. All pBAC™, a transcriptional enhancer sequence (hr5). The pBAC™-1 DNA 10 µg 70003-3
pAcPIE1 and pTriEx™ transfer plasmids are pBAC-5, pBAC-6, and pBAC-11 transfer plasmids pBACgus-1 DNA 10 µg 70054-3
supplied as highly purified supercoiled DNA. are based on a modified gp64 tandem promoter
Please see the table in the overview earlier in that contains both an immediate early promoter
pBAC-2cp DNA
pBACgus-2cp DNA
10 µg
10 µg
70004-3
70049-3
3
this chapter for individual vector features. and a late promoter, for continued expression in
Complete sequences and maps are available on the late phase of infection. In addition, pBAC-5, pBAC-3 DNA 10 µg 70088-3
Novagen’s home page at www.novagen.com. pBAC-6, and pBAC-11 may be used as expres- pBACgus-3 DNA 10 µg 70089-3
cloning/expression region cloning/expression region
pBAC-5 DNA 10 µg 70222-3
P6.9 pBACgus-5 DNA 10 µg 70223-3 4
3 16
60 2 pBAC-6 DNA 10 µg 70224-3
9
16
pBACgus-6 DNA 10 µg 70225-3

29
gus
l ef- 2
pBAC-7 DNA 10 µg 70104-3

pBAC
5.4 kbp
pBACgus
7.4 kbp
ori pBACgus-7 DNA 10 µg 70106-3 5
f1 or
product listing continued on next page

i
i
or
60
3
Ap le f Ap
-2
f 1 or i
Early Expression for Glycoproteins, Secreted

6
sion plasmids in the absence of virus for tran-
Proteins and Highly Processed Proteins
sient expression assays in transfected insect
The pAcPIE1 and pBAC-5, pBAC-6 and cells, as well as for the isolation of stably-trans-
pBAC-11 transfer plasmids are designed for the fected insect cell lines expressing target proteins
expression of genes beginning soon after infec-
tion, rather than beginning around 24 hours after
if co-transfected with pIE1-neo. The pBAC-5, -6,
and -11 transfer plasmids encode His•Tag® and
7
infection as for polh-based expression (see fig- S•Tag™ sequences, and pBAC-11 also encodes a
ure below). These baculovirus expression vec- CBDcenD•Tag sequence. These peptide affinity
tors enable expression of target genes before tags facilitate purification, quantitation, and
virus-induced cytopathic effects compromise the detection of fusion proteins.
function of protein processing pathways, partic- continued on next page 8
polh promoter gp64 promoter
0 h 24 h 48 h 72 h 96 h M 0h 6 h 10 h 24 h 32 h 52 h 81 h 96 h M
kDa kDa
9
– 150 – 150
– 100 – 100
– 75 – 75
– 50 – 50
– 35 – 35 10
– 25 – 25
– 15 – 15
pBAC-3/BacVector-3000 pBAC-6/BacVector-3000
11
Production and secretion of SEAP protein
The secreted human placental alkaline phosphatase gene (SEAP) was cloned into the Nco I site of pBAC-3 and pBAC-6. Sf9 cells, in serum-
Appendices
free medium, were infected with BacVector-3000 recombinant viruses. Samples of the medium (10 µl) were taken at the indicated times
Indices
and analyzed by Western blot. Expression under polh control (pBAC-3/SEAP) was detectable from 24 h post-infection. Expression under
gp64 control (pBAC-6/SEAP) was detectable by 6 h post-infection, and increased through 52 h post-infection. SEAP was detected using
S-protein Alkaline Phosphatase Conjugate (Cat. No. 69598-3).

119
Novagen
Protein Expression
1 Baculovirus Transfer Plasmids continued
High Level Expression Under polh Promoter Baculovirus Surface Display of Target Product Size Cat. No.
Proteins
The pBAC™-1 through -3, and pBAC-7 pBAC™-8 DNA 10 µg 70105-3
through -10 transfer plasmids are designed for The pBACsurf-1 transfer plasmid is designed
2 high level expression of proteins under control for in-frame insertion of target genes between
pBACgus-8 DNA
pBAC-9 DNA
10 µg
10 µg
70107-3
70138-3
of the polh promoter during the very late phase the gp64 signal sequence and the mature protein
of infection. The pBAC-1 transfer plasmids are coding sequence (under the control of the polh pBACgus-9 DNA 10 µg 70140-3
designed for expression of proteins without promoter). Expressed fusion proteins, including pBAC-10 DNA 10 µg 70218-3
additional tags, while pBAC-2 transfer plasmids glycoproteins, are secreted and incorporated
pBACgus-10 DNA 10 µg 70219-3
3 include S•Tag™ and His•Tag® peptide
sequences for convenient detection and purifica-
onto the virion surface, anchored by the trans-
membrane pBAC-11 DNA 10 µg 70220-3
insert
tion of expressed proteins. polh promoter domain of gp64 pBACgus-11 DNA 10 µg 70226-3
The pBAC-3 and pBAC-10 transfer plasmids gp64
(5). Target pro-
pBAC4x-1 DNA 10 µg 70045-3
are designed for expression of secreted proteins. teins are dis-
In addition to the S•Tag and His•Tag sequences, played at the pBACgus4x-1 DNA 10 µg 70060-3
3
60
4
162
they contain the 21 amino acid gp64 signal pep- poles of the viri- pBACsurf-1 DNA 10 µg 70055-3
le f - 2
9
tide sequence, which is capable of directing high on, where they
pBACsurf-1 pTriEx™-1.1 DNA 20 µg 70840-3
ORF10
levels of protein into the secretory pathway in appear to be
infected insect cells. The pBAC-10 transfer plas- localized as het- pTriEx-2 DNA 20 µg 70826-3
fi o
mids also include an N-terminal CBDcenD•Tag ero-oligomers pTriEx-3 DNA 20 µg 70823-3

ri
i
or
5 sequence.
CBD•Tag for Purification and Immobilization

Ap with the wild-type
gp64 also encod-
ed by the virus
pTriEx-4 DNA 20 µg 70824-3
target protein
of Target Proteins
(5). Additional Information Available
The pBAC-7 through -11 transfer plasmids For virus dis- BacVector Transfection Kits Protocol TB216
encode CBD•Tag™ (cellulose binding domain) play, inserts must Using pAcPIE1 Transfer Plasmids TB177
6 sequences for easy, low cost affinity purification.
The pBAC-10 and pBAC-11 transfer plasmids
BacVector recombinant
lack an internal
stop codon and
inNovations
Nos. 4a, 7, 8, 10
www.novagen.com
combine the gp64 secretion signal peptide and virus maintain the Patent and Licensing Information p. 278
the CBDcenD sequence to enable purification of appropriate open
secreted CBDcenD•Tag fusion proteins directly reading frame. Alternatively, target proteins may
from the culture medium. Purification is as sim- be displayed on the cell surface and on budded
7 ple as mixing the culture medium with CBIND™
Resin, washing, and eluting. We recommend use
virions if they remain in-frame with the down-
stream gp64 gene. Inserts in-frame with the N-
of BacVector®-3000 Triple Cut Virus DNA for terminal gp64 signal sequence and carrying an
making baculovirus constructs with CBD•Tag internal stop codon can produce target proteins
transfer plasmids. This virus has been engi- that may be secreted from the recombinant
neered to remove the chitinase gene whose virus-infected cell, provided they lack a mem-
8 product can be co-purified with target proteins brane-spanning sequence.
on CBIND Resins.
Multisystem Expression with pTriEx™
Multiple Gene Expression with One
Vectors
Recombinant Virus
9 The pBAC4x vectors are designed for co-

expression of up to 4 genes in the same cell, dur-
The pTriEx-1.1–4 vectors are compatible
with BacVector Triple Cut Virus DNA for con-
ing the very late phase of infection. These vec- struction and high level expression of recombi-
tors are extremely useful for the expression of nant baculoviruses. Expression is driven from
multisubunit proteins, multiple copies of a gene, the very late p10 promoter, and the vectors offer
multiprotein complexes, and for studies of pro- a choice of fusion tag configurations. See
10 tein:protein interactions (2–4). The pBAC-4x vec-
tors include two copies of the polh promoter and
Multisystem Expression later in this chapter for
additional information.
two copies of the p10 promoter.
1. Jarvis, D. L., Weinkauf, C. and Guarino, L. A. (1996)
inNovations 5, 1–5.
2. Weyer, U., and Possee, R. D. (1991) J. Gen. Virol.

11 72, 2967–2974.
3. Belyaev, A. S., and Roy, P. (1993) Nucleic Acids
Res. 21, 1219–1223.
4. Belyaev, A. S., Hails, R. S., and Roy, P. (1995) Gene
Appendices
156, 229–233.
Indices
5. Boublik, Y., Di Bonito, P., and Jones, I. M. (1995)

Bio/Technology 13, 1079–1084.

Novagen
Protein Expression
pBAC™ E. coli LIC Vector Kits Product

pBAC™-2cp E. coli
Size Cat. No.
1
Efficient directional cloning of PCR products into pBAC plasmids Ek/LIC Vector Kit 20 rxn 70021-3
pBAC™ E. coli LIC Vector Kits are designed Vectors, 100% of the vector-encoded N-terminal pBACgus-2cp E. coli
for convenient and highly efficient ligation-inde- fusion sequences can be removed from the puri- Ek/LIC Vector Kit 20 rxn 70051-3
pendent cloning of target genes into pBAC fied protein with either enterokinase or Factor pBAC-7 E. coli
Ek/LIC or Xa/LIC transfer plasmids in E. coli,
starting with inserts amplified with appropriate
Xa.
The kits provide necessary reagents for cre-
Ek/LIC Vector Kit
pBAC-8 E. coli
20 rxn 70116-3
2
primers (see pET LIC Vector Kits earlier in this ating single stranded overhangs, annealing with Xa/LIC Vector Kit 20 rxn 70117-3
chapter for details on the LIC method). A choice LIC Vector, and transforming NovaBlue Components:
of vectors with different fusion tag configura- Singles™ Competent Cells. A Control Insert is • 1 µg Linearized, prepared Ek/LIC or
tions is available. With all Ek/LIC and Xa/LIC included to verify performance. Xa/LIC Vector
• 8 µl Ek/LIC b-Gal or Ek/LIC GUS or
Xa/LIC b-Gal Control Insert
3
• 25 U T4 DNA Polymerase, LIC-qualified
pBAC-2cp Ek/LIC
pBACgus-2cp Ek/LIC • 50 µl 10X T4 DNA Polymerase Buffer
• 100 µl 100 mM DTT
Bpu1102 I
thrombin site enterokinase site
BamH I
Hind III
EcoR I
• 25 µl 25 mM dATP or dGTP
Sma I
Sac II
Nco I
Nhe I
Sph I
Xho I
Avr II
Sac I
Eag I
Not I
4
Stu I
Srf I
polh His•Tag S•Tag LIC LIC His•Tag

• 50 µl 25 mM EDTA
• 1.5 ml Nuclease-free Water
pBAC-7 Ek/LIC • 22 × 50 µl NovaBlue Singles™ Competent
Bpu1102 I
thrombin site enterokinase site Cells
BamH I
Hind III
EcoR I
SexA I
Sma I
Sac II
Nhe I
Sph I
Xho I
Avr II
Sac I
Eag I
Not I
Stu I
• 4 × 0.2 ml SOC Medium

Srf I
polh CBDclos•Tag S•Tag LIC LIC His•Tag

• 10 µl Test Plasmid
pBAC-8 Xa/LIC Available separately: 5
Bpu1102 I
thrombin site Factor Xa site

BamH I
Hind III
EcoR I
SexA I
Sac II
Nhe I
Sph I
Xho I
Avr II
Sac I
Eag I
Not I
Stu I
polh CBDclos•Tag S•Tag LIC LIC His•Tag

pBAC-2cp Ek/LIC
Vector 1 µg 70020-3
(linearized vector)
03
6
pBACgus-2cp Ek/LIC
6
16
29
lef -2
(linearized vector)
pBAC-2cp
pBAC-7 pBAC-7 Ek/LIC
f1 or
pBAC-8 (linearized vector)

i
pBAC-8 Xa/LIC
or
Ap Vector
(linearized vector)
1 µg 70109-3 7
NovaBlue Singles™ 11 rxn 70181-3
P6.9 Competent Cells 22 rxn 70181-4
16
2
T4 DNA Polymerase,
9

8
gus
ori
pBACgus-2cp
Ek/LIC Vector Kits Manual TB163
Xa/LIC Vector Kits Manual TB184
60
3
le f Ap Vector Cloning Region Sequences Appendix
-2
f 1 or i
inNovations
www.novagen.com
Nos. 4a, 7 9
10
11
Appendices
Indices

121
Novagen
Protein Expression
1 Insect Cell Transient and Stable Expression Plasmids

Vectors for continuous and/or transient expression in insect cells
pIE1 and pAcPIE1 Plasmids secretory pathway glycoproteins, cell surface Product Size Cat. No.
receptors and basic cellular processes like apop-
The pIE1 plasmids are designed for the pIE1-1 DNA 10 µg 69088-3
tosis (6).
expression of foreign genes under the control of
2 the immediate early baculovirus promoter, ie1, pBAC Plasmids
pIE1-2 DNA
pIE1-3 DNA
10 µg
10 µg
69089-3
69090-3
in stably-transformed insect cells (1–3). The
The pBAC™-5, -6, and -11 transfer plasmids
pAcPIE1 plasmids carry the same ie1 promoter pIE1-4 DNA 10 µg 69091-3
drive transcription of target genes by the cellular
that is in the pIE1 plasmids. Although the pIE1-neo DNA 10 µg 70171-3
RNA polymerase from the early element of the
pAcPIE1 transfer plasmids can be used to gener-
gp64 promoter. These vectors are therefore suit- pAcP(+)IE1-1 DNA 10 µg 69092-3
3 ate recombinant baculoviruses, they may also be
used for direct transfection. To establish stable
able for continuous or transient expression by pAcP(+)IE1-2 DNA 10 µg 69093-3
transfection with or without the pIE1-neo selec-
transformants, insect cells are co-transfected pAcP(+)IE1-3 DNA 10 µg 69094-3
tion plasmid, respectively. Higher expression lev-
with the pIE1 recombinant plasmid and the
els can be obtained with these plasmids than pAcP(+)IE1-4 DNA 10 µg 69095-3
pIE1-neo selection plasmid (4, 5) and selected
with the ie1 promoter plasmids described above. pAcP(–)IE1-5 DNA 10 µg 70170-3
in the presence of G418. For faster results, tran-
4 sient expression can be achieved by transfection
in the absence of the selection plasmid. Efficient
1. Jarvis, D. L., Fleming, J. G. W., Kovacs, G. R.,
Summers, M. D., and Guarino, L. A. (1990)
pAcP(–)IE1-6 DNA 10 µg 69097-3
Bio/Technology 8, 950–955. pBAC™-5 DNA 10 µg 70222-3
transfection of Sf9 cells is obtained using
2. Jarvis, D. L. (1993) Insect Cell Culture Engineering, pBACgus-5 DNA 10 µg 70223-3
Eufectin™ Transfection Reagent.
(Goosen, M. F. A., Daugulis, A., and Faulkner, P.,
The transformants produce low levels of tar- eds), pp. 193–217, Marcel Dekker, Inc., New York. pBAC-6 DNA 10 µg 70224-3
5 get protein constitutively without the need for

baculovirus infection (1). These cells can pro-
cess secretory pathway glycoproteins more effi-
3. Jarvis, D. L., and Guarino, L. A. (1995) “Baculovirus
Expression Protocols” in Methods in Molecular
Biology, Vol 39, (Richardson, C. D., ed),
pBACgus-6 DNA
pBAC-11 DNA
10 µg
10 µg
70225-3
70220-3
pp. 187–202, Humana Press, Clifton, N. J.
ciently than baculovirus-infected insect cells pBACgus-11 DNA 10 µg 70226-3
4. Wigler, M., Sweet, R., Sim, G. K., Wold, B., Pellicer,
because there are no virus-induced cytopathic Eufectin™ Transfection
A., Lacy, E., Maniatis, T., Silverstein, S., and Axel, R.
effects. In addition, insect cells lack certain cell (1979) Cell 16, 777–785. Reagent 30 µl 70035-3
6 surface molecules that can contribute to back-
ground in mammalian-based receptor screening
5. Colbere-Garapin, F., Horodniceanu, F., Kourilsky,
P., and Garapin, A. C. (1981) J. Mol. Biol. 150, 1–14.
G 418 Sulfate,
Cell Culture Tested
100 mg
250 mg
345810
345810
systems. These advantages make pIE1-based 6. Cartier, J. L., Hershberger, P. A., and Friesen, P. D. 500 mg 345810
expression in stably-transformed insect cells an (1994) J. Virol. 68, 7728–7737. 1g 345810
5g 345810
excellent choice for many studies, including
G 418 Sulfate, 10 ml 345812
7 Insect Vectors for Transient and Stable Expression
Sterile-Filtered
Aqueous Solution,
20 ml
50 ml
345812
345812
Cell Culture Tested
Vector Provides ATG MCS Orientation Special Features/Applications Additional Information Available
pIE1-1 yes BsaB I–BamH I Low level continuous expression in Transfection with pIE1-neo TB176
8 pIE1-2
pIE1-3
yes
no
BamH I–BsaB I
BsaB I–BamH I
transfected insect cells. Vector Cloning Region Sequences
Appendix
www.novagen.com
pIE1-4 no BamH I–BsaB I inNovations No. 5
pAcP(+)IE1-1 yes Bgl II–Pme I Very early low level expression for complete
pAcP(+)IE1-2 yes Pme I–Not I processing. Vectors differ in providing
pAcP(+)IE1-3 no Bgl II–Pme I vector-encoded ATG and selection of cloning
9 pAcP(+)IE1-4
pAcP(–)IE1-5
no
yes
Pme I–Not I
BamH I–Bgl II
sites.
Very early low level expression, plus
NOTE: Products shown
pAcP(–)IE1-6 no BamH I–Bgl II compatibility with larval infection. in red will be
pIE1-neo NA NA Selectable marker for cotransfection discontinued
pBAC-5, pBACgus-5
pBAC-6, pBACgus-6
yes
yes
Sma I–Xho I
Sma I–Xho I
Options for His•Tag®/S•Tag™/CBD•Tag™
fusions, signal sequences for secretion,
effective May 1, 2003.
10 pBAC-11, pBACgus-11 yes Srf I–Xho I Tb/Ek sites, mid-level expression
11
Appendices
Indices

Novagen
Protein Expression
FastPlax™ Titer Kit Product Size Cat. No.

1
Rapid, reliable titering of baculovirus stocks FastPlax™ Titer Kit 5 assays 70850-3
Components:
The FastPlax™ Titer Kit is designed for monoclonal antibody against gp64, which is • 50 ml 10X TBST
determination of baculovirus titers in just 24 to added directly to fixed cells. Antibody binding is • 6 ml 10% Gelatin
48 hours instead of 3 to 4 days as in other meth- detected with Goat Anti-Mouse IgG b- • 10 µl FastPlax Antibody
ods. Classical determination of baculovirus titer
has relied on the appearance of plaques, but the
Galactosidase Conjugate followed by enhanced
color development with X-Gal/NBT substrates.
• 300 µl Goat Anti-Mouse IgG b-Galactosidase
Conjugate
2
FastPlax Kit takes advantage of the appearance Plaques are clearly distinguished by their dark • 300 µl NBT
of detectable levels of AcNPV gp64 glycoprotein blue color. The kit contains sufficient reagents • 300 µl X-Gal
on the cell surface as early as 8 to 24 hours post- to perform five 6-well plate assays. Available separately:
infection. Detection is based on a high affinity Product Size Cat. No.
FastPlax Antibody 10 µl 70814-3 3
FastPlax Titer Kit Protocol TB272
BacVector® Transfection Kits Protocol TB216
4
5
Detection of baculovirus-infected cells 24 hours
post infection using the FastPlax Kit
6
™
pBAC , pAcPIE1, and pIE1 Plasmid Primers
Convenient amplification and sequencing
pBAC, pAcPIE1, and pIE1 vector primers Product Applicable Vectors Size Cat. No.
have been developed for amplification and Upstream Primers 7
sequencing of recombinants. The T7/polh primer
is suitable for use with the Single Tube Protein®
T7/polh Primer pBAC™/pBACgus-1, 2cp, 3, 7, 8, 9, 10, surf1 100 pmol 70046-3
System 3 for synthesis of pBAC recombinant polh promoter Primer pBAC/pBACgus-1, 2cp, 3, 7, 8, 9, 10, surf1 500 pmol 70007-3
proteins from PCR products in vitro. See gp64 Signal Primer BAC-3, 6, 10, 11, surf-1 500 pmol 70008-3
Chapter 1, PCR for further information and for
primer sequences.
S•TagBAC Primer pBAC/pBACgus-2cp, 3, 5, 6, 7, 8, 10, 11 500 pmol 70009-3 8
CBDclos•Tag Primer pBAC/pBACgus-7, 8 500 pmol 70118-3
CBDcex•Tag Primer pBAC/pBACgus-9 500 pmol 70142-3
IE1 Promoter Primer all pIE1 and pAcPIE1 500 pmol 69103-3
gp64 Promoter Primer
T7/gp64 Promoter Primer
pBAC-5, 6, 11, surf1
pBAC-5, 6, 11, surf1
500 pmol
100 pmol
70242-3
70217-3
9
Downstream Primers
1629DWN Primer all pBAC plasmids except pBACsurf-1 500 pmol 70011-3
ASgp64 Primer pBACsurf-1 500 pmol 70062-3
AS603UTR Primer pBAC4x-1 500 pmol 70063-3 10
ASbasicprom Primer pBACgus4x-1 500 pmol 70064-3
AS CBDcex Primer pBAC/pBACgus-9 500 pmol 70143-3
HRAS Primer all pIE1 500 pmol 69104-3
TV14AS Primer pAcP(+)IE1-1, -2, -3, -4 500 pmol 69105-3 11
TV56AS Primer pAcP(–)IE1-5, -6 500 pmol 69106-3
Appendices
Indices

123
Novagen
Protein Expression
Prokaryotic
Expression
1 Sf9 Insect Cells Product Size Cat. No.
Serum-free adapted Sf9 cells Sf9 Insect Cells 3 vials 71104-3
Novagen’s Sf9 Insect Cells are provided as cells. Cells are

frozen stocks of Spodoptera frugiperda Sf9 cells shipped on dry ice.
Sf9 Insect Cells Protocol TB329
for establishment of cultures suitable for any Upon receipt, the
2 baculovirus application. These cells plus
BacVector® Insect Cell Medium are recommend-
vials should be
removed from their
ed for co-transfection of transfer plasmids with container and either
BacVector Triple Cut Virus DNA for the con- recovered immedi-
struction of baculovirus recombinants. After ately, or placed at
recovery, they can be grown as semi-adherent –70°C and used within two weeks. For longer
3 cultures in tissue culture flasks or in suspension term storage, place in liquid nitrogen.
as shaker cultures. Each vial contains 2 × 106
4
BacVector® Insect Cell Medium Product
®
Size Cat. No.
Serum-free medium optimized for growth of Sf9 Insect Cells BacVector Insect
Cell Medium 1 liter 70590-3
5 BacVector® Insect Cell Medium is optimized

for serum-free growth of Sf9 Insect Cells. This
cell/medium combination is recommended for
binant baculoviruses, and is compatible with
Eufectin™ Transfection Reagent. This medium
is also recommended for plaque assays using the
Sf9 Insect Cells Protocol TB329
co-transfection of BacVector Triple Cut Virus agarose overlay technique. Store in the dark at BacVector Transfection Kits Protocol TB216
DNA and transfer plasmids to construct recom- 4°C.
TriEx™ Sf9 Cells Product Size Cat. No.
7 Serum-free adapted Sf9 derivative for high-yield protein expression TriEx™ Sf9 Cells 3 vials 71023-3
The unique TriEx™ Sf9 Cells are derived as shaker cultures.

from a high-yielding clone of Sf9 cells. Pre-adapt- Each vial contains
TriEx Sf9 Cells Protocol TB314
ed for growth in Novagen’s serum-free TriEx 2 × 106 cells. Cells
Insect Cell Medium, these cells are recommend- are shipped on dry inNovations No. 12
8 ed for superior growth and protein yields by ice. Upon receipt,
either baculovirus infection or transfection of the vials should be
appropriate vectors. For co-transfection of plas- removed from their
mids with linearized baculovirus DNA, we rec- container and either recovered immediately, or
ommend Sf9 Insect Cells and BacVector Insect placed at –70°C and used within two weeks. For
9 Cell Medium. After recovery from the frozen

stock, the cells can be grown as semi-adherent
longer term storage, place in liquid nitrogen.
cultures in tissue culture flasks or in suspension
10
TriEx Insect Cell Medium Product Size Cat. No.
Serum-free medium optimized for growth of TriEx Sf9 Cells TriEx™ Insect Cell
Medium 1 liter 71022-3
TriEx Insect Cell Medium is a serum-free based on rapid, vigorous cell growth and high
11 medium optimized for growth and protein pro-
duction from TriEx Sf9 Cells. This matched
protein expression levels. Store in the dark at
4°C.
cell/medium combination has been selected inNovations No. 12
Appendices
Indices

Novagen
Protein Expression
Eufectin™ Transfection Reagent Product Size Cat. No.

1
Efficient transfection of Sf9 cells Eufectin™ Transfection
Reagent 30 µl 70035-3
Eufectin™ Transfection Reagent is a lipid- the BacVector Transfection Kits protocol. The
based formulation optimized for transfection of reagent is also suitable for transfection of plas- Additional Information Available
insect cells with minimal cytotoxicity. Each lot mids into Sf9 cells for direct expression from
of Eufectin is qualified for efficient co-transfec-
tion of BacVector® Triple Cut Virus DNA and a
insect promoters. Sf9 Insect Cells Protocol
inNovations
TB329
No. 4a
2
pBAC transfer plasmid into Sf9 insect cells using
BacPlaque™ Agarose Product Size Cat. No.

4
Qualified agarose for optimal plaque assays BacPlaque™ Agarose 30 g 70034-3
Agarose overlays are commonly used in bac- acteristics required to obtain good overlays.
ulovirus plaque assays. Many commercial “DNA Novagen’s BacPlaque™ Agarose is pre-qualified
grade” agaroses either contain contaminants that
are toxic to insect cells or lack the physical char-
in plaque assays and transfections to ensure con-
sistent performance.
BacPlaque Agarose Protocol
inNovations
TB156
No. 4a 5
X-Gluc Solution Product Size Cat. No. 7

A colorimetric stain for GUS activity X-Gluc Solution 500 µl 70036-3
The b-glucuronidase substrate X-Gluc (5- BacVector GUS Recombinant Virus. It is provid-
bromo-4-chloro-3-indolyl-b-D-glucuronide) is a ed as a 20 mg/ml stock solution, ready for dilu-
colorimetric stain for GUS activity, and is used
to identify recombinants containing pBACgus
tion and use in staining plaques for GUS activity.
Each vial contains enough substrate to stain 15
inNovations No. 4a 8
vectors, the LIC GUS Control Insert, and plates.
10
BacVector GUS recombinant plaque stained with
Neutral Red and X-Gluc (100X magnification)
11
Appendices
Indices

125
Novagen
Protein Expression
Mammalian Expression
1 Mammalian Expression Introduction

Versatile pTriEx™ vectors for mammalian expression
Many mammalian proteins undergo various Furthermore, mammalian cells are well suited expression in mammalian cells. In addition,
types of post-translational processing and modi- for various types of recombinant protein studies these unique vectors incorporate the appropriate
fications. In order to express active recombinant such as functional activity assays and physiologi- signals for high-level expression of proteins in
2 mammalian proteins that are properly post-trans-
lationally processed and modified, mammalian
cal effects on cellular functions. Novagen’s
pTriEx™ vectors feature strong transcription
bacteria and insect cells.
cell lines are the best choice as expression hosts. and translation signals for optimal protein
3 pTriEx™ Transient Expression Vectors Product Size Cat. No.
High-level transient expression in mammalian cells pTriEx™-1.1 DNA 20 µg 70840-3

pTriEx Multisystem Expression Vectors tiation signals include a ribosome binding site
enable high-level “broad-spectrum” transient for bacterial expression followed by an optimal
4 expression in vertebrate cells driven by either an
actin promoter (CAG) or CMV promoter, along
consensus sequence for mammalian cell expres-
sion. Other common features include blunt cut- TriEx™UP Primer 500 pmol 70846-3
with CMVie enhancer and rabbit β-globin ting restriction enzyme sites in every open read-
TriExDOWN Primer 500 pmol 70847-3
polyadenylation signals. RNA generated from the ing frame (ORF) at both ends of MCS to facili-
CAG or CMV promoter possesses an intron tate in-frame cloning, ampicillin resistance mark-
Product Cat. No.
designed to facilitate mRNA processing and er, pUC replication origin for superior plasmid
5 export. In addition, all pTriEx vectors contain
the tightly controlled T7lac promoter for
yield, choice of N-terminal His•Tag®/S•Tag™ or
C-terminal HSV•Tag®/His•Tag sequences for
pTriEx-1.1 Cloning Kit
pTriEx-2 Cloning Kit
70898-3
70866-3
inducible expression in E. coli, and p10 bac- detection and purification, and protease cleav- pTriEx-3 Cloning Kit 70873-3
ulovirus promoter for high level expression in age sites for effective removal of N-terminal
pTriEx-4 Cloning Kit 70879-3
baculovirus-infected insect cells. Translation ini- fusion tags.
Components:
6 Fusion Tags Protease • 20 µg pTriEx Vector
Cleavage • 55 µl Clonables™ 2X Ligation Premix
Vector Promoter N-terminal C-terminal Sites Special Features/Applications • 10 µl Clonables Positive Control
pTriEx-1.1 T7lac, p10, none HSV•Tag® none Optional C-terminal tags for detection and
b-actin His•Tag® purification
Cells
7 pTriEx-2 T7lac, p10,
b-actin
His•Tag
S•Tag™
HSV•Tag
His•Tag
thrombin N-terminal tags for detection and purification
enterokinase Homogeneous FRET assay
• 2 × 2 ml
• 20 µl
SOC Medium
Test Plasmid
pTriEx-3 T7lac, p10, none HSV•Tag none Optional C-terminal tags for detection and Available separately:
CMV His•Tag purification
pTriEx-4 T7lac, p10, His•Tag HSV•Tag thrombin N-terminal tags for detection and purification
CMV S•Tag His•Tag enterokinase Homogeneous FRET assay
GeneJuice™ 1 ml 70967-3
8 Transfection Reagent 10 ml
0.3 ml
70967-4
70967-5
pTriEx-1.1 pTriEx-2
CMV ie promoter pTriEx-3 pTriEx-4 Additional Information Available
T7lac T7lac TriEx System Manual TB250
CMV ie enhancer Chicken ß-actin promoter Vector Cloning Region Sequences Appendix
9 3
p10
Nco I
p10
Nco I
60 Exon 1
intron EcoR V His•Tag
Sma I Xcm I
2
Ecl136 I S•Tag
le f-
BamH I thrombin site

Exon 2
EcoR I Sma I
Bgl II enterokinase site
Asc I PshA I
10 Rabbit ß-globin terminator

T7 terminator
BssH I*
Pst I
Sse8387 I
BseR I
EcoR V
Ecl136 I
Ap
Kpn I BamH I
PinA I EcoR I
29
16 Nsp V Bgl II
Hind III Asc I
o ri Not I BssH I*
Eag I* Pst I
Pvu II Sse8387 I
11 actin promoter
pTriEx-1.1
pTriEx-2
CMV promoter
pTriEx-3
pTriEx-4
Bst1107 I
Pml I
HSV•Tag
Kpn I
PinA I
Nsp V
Xho I Hind III
His•Tag Not I
Dra III Eag I*
Bsu36 I Pvu II
Appendices
Bst1107 I
* not unique in pTriEx-1.1 or pTriEx-2 Pml I
Indices
HSV•Tag
Xho I
His•Tag
Dra III
Bsu36 I

Novagen
Protein Expression
pTriEx™ Transient Expression Vectors continued

1
His•Tag fusion protein detection Nuclear staining
3
A B
Expression of a pTriEx His•Tag fusion protein in transiently transfected COS-1 cells

pTriEx™ plasmid DNA encoding a His•Tag firefly luciferase (Fluc) fusion protein was transiently transfected into COS-1 cells with GeneJuice™
Transfection Reagent. Twenty-four hours after transfection, cells were fixed, blocked with BSA and horse serum, and then exposed to His•Tag® 4
Monoclonal Antibody (1:1000 dilution of 0.2 mg/ml) followed by a Cy3 conjugated Goat Anti-Mouse IgG. Hoechst 33258 was used for visualiza-
tion of cell nuclei. A, Fluorescent staining of His•Tag Fluc; B, Hoechst staining of the same field as in A showing both transfected and non-
transfected cells.
5
pTriEx™-4 Ek/LIC Vector Kit Product Size Cat. No.
Efficient directional cloning of PCR products into pTriEx-4 pTriEx™-4 Ek/LIC

The pTriEx™-4 Ek/LIC Vector Kit is designed N-terminal fusion sequences can be removed Components:
6
for convenient and highly efficient ligation-inde- from the purified protein with enterokinase. • 1 µg pTriEx-4 Ek/LIC Vector
pendent cloning (LIC) of target genes into the The kit provides necessary reagents for cre- • 8 µl Ek/LIC Control Insert
versatile pTriEx-4 vector, starting with inserts ating single stranded overhangs, annealing with • 25 U T4 DNA Polymerase, LIC qualified
amplified with appropriate primers (see pET LIC LIC Vector, and transforming NovaBlue • 50 µl 10X T4 DNA Polymerase Buffer
Vector Kits earlier in this chapter for details on
the LIC method). When inserts are properly
Singles™ Competent Cells. A Control Insert is
included to verify performance.
• 100 µl
• 25 µl
100 mM DTT
25 mM dATP or dGTP
7
cloned into the Ek/LIC site, the vector-encoded • 50 µl 25 mM EDTA
Cells
pTriEx-4 Ek/LIC thrombin site

• 4 × 0.2 ml SOC Medium
8
Sse8387 I
Bst1107 I
enterokinase site • 10 µl Test Plasmid

Bsu36 I
BamH I
EcoR V
Hind III
EcoR I
Nsp V
PinA I
Dra III
Sma I
Xcm I
Pvu II
Nco I
Kpn I
Bgl II
Xho I
Pml I
Sac I
Asc I
Not I
Pst I
CMV T7lac polh His•Tag S•Tag LIC LIC HSV•Tag His•Tag

Rabbit ß-globin terminator
Exon 2 T7 terminator pTriEx-4 Ek/LIC
intron Vector 1 µg 70843-3
Exon 1
9
16
(linearized vector)
29
CMV promoter
NovaBlue Singles™ 11 rxn 70181-3
CMV ie enhancer
Competent Cells 22 rxn 70181-4
o ri
T4 DNA Polymerase,
lef-2

03 10
6
Ap Additional Information Available

Ek/LIC Vector Kits Manual TB163
11
Appendices
Indices

127
Novagen
Protein Expression
1 pTriEx™ Stable Expression Vectors Product Size Cat. No.
Efficient selection of stable mammalian cell lines expressing genes of interest pTriEx™-1.1 Hygro
DNA 20 µg 70928-3
Novagen’s pTriEx™ stable expression vec- respectively. Translation initiation signals pTriEx-1.1 Neo
tors are designed to efficiently select mammalian include a ribosome binding site for bacterial DNA 20 µg 70927-3
cell lines expressing target genes. This is expression followed by an optimal consensus pTriEx-2 Hygro
2 achieved by translation of both the gene of inter-
est and a downstream selective marker gene
sequence for vertebrate cell expression (3).
Blunt cutting restriction enzyme sites in every
DNA
pTriEx-2 Neo
20 µg 70930-3
from a single messenger RNA, which ensures open reading frame (ORF) are incorporated at DNA 20 µg 70929-3
that all drug-resistant cells also produce the tar- both ends of the MCS to facilitate in-frame pTriEx-3 Hygro
get protein. Translation of the selective marker cloning. All of the vectors encode optional DNA 20 µg 70932-3
gene is controlled by an EMCV-derived Cap- HSV•Tag® and His•Tag® sequences at the distal
3 Independent Translation Enhancer (CITE, or end of the MCS to enable the construction of C-
pTriEx-3 Neo
DNA 20 µg 70931-3
IRES, internal ribosome entry site; 1, 2). After terminally-tagged fusion proteins. The pTriEx-2
pTriEx-4 Hygro
selection with neomycin or hygromycin, virtually and -4 versions also encode N-terminal His•Tag DNA 20 µg 70934-3
all surviving cells exhibit stable high level and S•Tag™ sequences followed by thrombin
pTriEx-4 Neo
expression of target gene, eliminating the need and enterokinase cleavage sites. The promoter DNA 20 µg 70933-3
4 to screen a large number of surviving colonies.
The pTriEx CITE-based stable expression
and cloning regions are flanked on each side by
segments of baculovirus genomic DNA that facil- TriExUP Primer 500 pmol 70846-3
vectors contain the following expression ele- itate the generation of baculovirus recombinants TriExDOWN Primer 500 pmol 70847-3
ments: the pTriEx tri-promoter cassette, a multi- at the viral polh locus.
cloning site (MCS), the IRES (CITE), the antibi- The pTriEx stable expression vectors are Available separately:
otic resistance gene (either neomycin phospho- optimally transfected using GeneJuice™ Product Size Cat. No.
5 transferase, NPTII, or hygromycin B phospho-

transferase, HPH), and the rabbit b-globin
Transfection Reagent for high efficiency with
minimal cytotoxicity for many cell lines.
GeneJuice™
Transfection Reagent
1 ml
10 ml
70967-3
70967-4
0.3 ml 70967-5
polyadenylation signal.
1. Jang, S. K., Krausslich, H., Nicklin, M. J. H., Duke, G.
All of the multisystem expression features of M., Palmenberg, A. C. and Wimmer, E. (1998) J.
the pTriEx vectors are maintained in the selec- Virol. 62, 2636–2643. Additional Information Available
tive marker vectors. The b-actin promoter (CAG) 2. Jackson, R. J., Howell, M. T., and Kaminski, A.
6 or CMVie promoter followed by T7lac and p10 (1990) Trends Biochem. Sci. 15, 477–483.
TriEx System Manual
TB250
www.novagen.com
Appendix
promoters are used to direct the high level 3. Kozak, M. (1989) J. Cell Biol. 108, 229–241.
expression of target genes in vertebrate cells, E. continued on next page
coli, and baculovirus-infected insect cells,
9
Stable expression of b-galactosidase in BHK cells transfected with a pTriEx-1.1 Neo recombinant
The β-galactosidase gene from E. coli was cloned into pTriEx-1.1 Neo. The resulting plasmid was transfected into BHK cells by using GeneJuice™
Transfection Reagent. Forty-eight hours post-transfection, neomycin (500 µg/ml) was added to the culture medium. After 7 days of further cul-
10 ture, surviving cells were trypsinized, seeded into a new T-75 flask and grown in the presence of 1000 µg/ml neomycin for an additional 2 weeks.
To assess expression of the b-galactosidase target gene, cells were stained with X-gal substrate using a standard protocol. The left and right
panels are photographs of the same area in low and high magnification, respectively. Virtually all of the cells in the polyclonal population exhibit-
ed high levels of b-gal expression.
11
Appendices
Indices

Novagen
Protein Expression
pTriEx™ Stable Expression Vectors continued

1
Fusion Tags
Protease
Vector Promoters N-terminal C-terminal Cleavage Sites Special Features
pTriEx-1.1 Hygro T7lac, p10, none HSV•Tag® none Optional C-terminal tags 2
b-actin His•Tag® Hygromycin selection
pTriEx-1.1 Neo T7lac, p10, none HSV•Tag none Optional C-terminal tags
b-actin His•Tag Neomycin selection
pTriEx-2 Hygro T7lac, p10, His•Tag HSV•Tag thrombin N-terminal tags
b-actin S•Tag His•Tag enterokinase Homogeneous FRET assay
Hygromycin selection
3
pTriEx-2 Neo T7lac, p10, His•Tag HSV•Tag thrombin N-terminal tags
b-actin S•Tag His•Tag enterokinase Homogeneous FRET assay
Neomycin selection
pTriEx-3 Hygro T7lac, p10, CMV none HSV•Tag none Optional C-terminal tags
His•Tag Hygromycin selection
4
pTriEx-3 Neo T7lac, p10, CMV none HSV•Tag none Optional C-terminal tags
His•Tag Neomycin selection
pTriEx-4 Hygro T7lac, p10, CMV His•Tag HSV•Tag thrombin N-terminal tags
S•Tag His•Tag enterokinase Homogeneous FRET assay
Hygromycin selection
pTriEx-4 Neo T7lac, p10, CMV His•Tag
S•Tag
HSV•Tag
His•Tag
thrombin
enterokinase
N-terminal tags
Homogeneous FRET assay
5
Neomycin selection
pTriEx-1.1 Hygro
pTriEx-1.1 Neo
pTriEx-2 Hygro
pTriEx-2 Neo
pTriEx-3 Hygro
pTriEx-3 Neo
pTriEx-4 Hygro
pTriEx-4 Neo
6
T7lac T7lac T7lac T7lac
lef-2 lef-2
Ap 60
3
p10 p10
Ap 60
3
p10 p10
Nco I Nco I Nco I Nco I
CMV ie enhancer
EcoR V
Sma I
Ecl136 I
His•Tag
Xcm I
S•Tag CMV ie enhancer
EcoR V
Sma I
Ecl136 I
His•Tag
Xcm I
S•Tag
7
o ri
o ri
Chicken ß-actin promoter BamH I thrombin site BamH I thrombin site

CMV ie promoter
EcoR I Sma I EcoR I Sma I
Exon 1 Bgl II enterokinase site Exon 1 Bgl II enterokinase site
intron Asc I PshA I intron Asc I PshA I
BseR I BseR I
1 62 9
1 62 9
Pst I* BssH II
Sse8387 I EcoR V Pst I* EcoR V
PinA I †
2
Ecl136 I Sse8387 I Ecl136 I

on
on
T7 terminator T7 terminator
Nsp V BamH I PinA I † BamH I
8
Ex
Ex
Rabbit ß-globin terminator Not I EcoR I Rabbit ß-globin terminator Nsp V EcoR I
E Pvu II Bgl II E Not I Bgl II
h y g ro CIT Bst1107 I Asc I h y g ro CIT Pvu II Asc I
or neo HSV•Tag Pst I* or neo Bst1107 I BssH II
Xho I Sse8387 I HSV•Tag Pst I*
His•Tag PinA I † Xho I Sse8387 I
Bsu36 I Nsp V His•Tag PinA I †
Not I Bsu36 I Nsp V
Pvu II Not I
* not unique in pTriEx-1.1 Neo or pTriEx-2 Neo
†
not unique in pTriEx-1.1 Hygro or pTriEx-2 Hygro
Bst1107 I
HSV•Tag
Xho I
His•Tag
* not unique in pTriEx-3
†
Neo or pTriEx-4 Neo
not unique in pTriEx-3 Hygro or pTriEx-4 Hygro
Pvu II
Bst1107 I
HSV•Tag
Xho I
9
Bsu36 I His•Tag
Bsu36 I
10
11
Appendices
Indices

129
Novagen
Protein Expression
1 GeneJuice™ Transfection Reagent Product Size Cat. No.
High efficiency transfection of mammalian cells GeneJuice™ 1 ml 70967-3

Transfection Reagent 10 ml 70967-4
0.3 ml 70967-5
GeneJuice™ Transfection
Reagent is a proprietary for-
mulation optimized for max- Additional Information Available
2 imal transfection efficiency,

ease of use, and minimal
GeneJuice Transfection Reagent Protocol TB289
cytotoxicity. This transfec-

tion reagent is a superior alter-
native to a wide variety of other techniques
including calcium phosphate coprecipitation,
3 electroporation, microinjection, biolistic particle
delivery, and complex formation with DEAE-
dextran.
Whereas many available transfection
reagents are based on cationic lipid formula-
No transfection Cationic lipid based reagent GeneJuice
4 tions, GeneJuice is composed of a nontoxic cel-
lular protein and a small amount of a novel Toxicity comparison
polyamine. The unique chemistry provides sev- Three replicate COS-7 cultures were left untreated, transfected with a popular cationic lipid based transfec-
eral advantages over lipid-based transfection, tion reagent, and transfected with GeneJuice according to recommended protocols. Cellular damage is visu-
including: alized by rounding up and detachment from the plate surface. The photographs, taken 48 h post transfec-
tion, show that GeneJuice caused much less cytotoxicity than the other reagent.
• Highly efficient DNA transfer for both stable
5 •
and transient transfections
Minimal cellular toxicity
120 5.0
• Compatibility with both serum-containing 4.5
100 COS-1 HeLa
and serum-free media 4.0
RLU/ml (x 10-3)
RLU/ml (x 10-3)
3
3.5
• Simple protocol—no need for media changes 80
6 • Ideal for high throughput (HT) transfection 60
3.0
2.5
in a multi-well plate format 2.0
40
GeneJuice Transfection Reagent has been 1.5
20 1.0
demonstrated to provide excellent performance
0.5
in both stable and transient transfection of 0 0
7 eukaryotic cells and is ideal for use with
GeneJuice
L2
GP
GS
GeneJuice
L2
GP
GS
Novagen’s pTriEx™ transient and stable expres-
sion vectors.
The 1 ml size provides enough reagent to
perform up to 500 transfections in standard 35 8.0 9.0
mm plates. The reagent is also available in an 8.0
8 introductory 0.3 ml size. GeneJuice is supplied
7.0 L6
7.0
NIH-3T3
RLU/ml (x 10-3)
6.0
RLU/ml (x 10-2)
as a ready-to-use sterile solution. 6.0

5.0
5.0
continued on next page 4.0 4.0
3.0 3.0
9 2.0
1.0
2.0
1.0
0
0
GeneJuice
L2
GP
GS
GeneJuice
L2
GP
GS
10 12.0 3.5
10.0 HepG2 3.0 Raw264.7

RLU/ml (x 10-2)
RLU/ml (x 10-2)
2.5
8.0
2.0
Transfection efficiency with GeneJuice vs. commonly used
11 competitor reagents
6.0
1.5
The indicated cell lines were plated at a density of 30,000 cells per well in 24-well plates the 4.0
1.0
day prior to gene delivery. Transfections and media changes were performed according to the
2.0 0.5
manufacturers’ optimized protocols. For transfection, 0.5 µg of UltraMobius™ purified
Appendices
pTriEx™-4 FLuc plasmid DNA was complexed with the relevant reagent and introduced into 0 0
Indices
each well. After 48 h, the cells were extracted with Reportasol™ Extraction Buffer and FLuc
GeneJuice
L2
GP
GS
GeneJuice
L2
GP
GS
activity was assayed. Data are represented as relative light units per milliliter of extract
(RLU/ml). All values reflect an average of four replicate cultures.

Novagen
Protein Expression
GeneJuice™ Transfection Reagent continued

1
Cell types successfully transfected with GeneJuice Transfection Reagent
10T1/2 BC3 CFPAC-1 HCT-116 IEC-6 Melanocyte PS-1 SW-837
3T3-L1
A204
BCBL
BHK-21
Chang Liver
CHO
HEK293
HeLa
JEG-3
Jurkat
MG-63
Neuroblastoma
R2C
RAW 264.7
T3M4
TM4 2
A431 C3H/10T1/2 COS-1 Hep 3B2.1-7 KB NPK RBL-2H3 U937
A549 C6 COS-7 HepG2 L57-3-11 NT2/D1 RMP-41 UCD
alpha TC1-6 C2C12 DDTI MF-2 Hepa 1-6 L-6 OV-1063 SC-1 Vero
AR 42J Caco-2 DT40 Ht-29 L-929 OVCAR3 Schneider line2 WE-38
As4.1 Caki-1 ECV304 HTB-37 MA-10 P4 SK-N-MC Primary aortic smooth
AtT-20
B50
Calpan-1
Calu-1
EL4
ES-E14TG2a
HTB-45
Huh-7
McA-RH7777
MCF-7
P19
PC12
SK-N-SH
SKOV3
muscle cells
Primary keratinocytes 3
BC-1 Calu-6 EVSCC17M HUVEC MCF-10-2A PA317 STO
BC-2 CCL-131 H9c2 IC21 MDCK PAM212 SW-480
COS-7 HeLa
GeneJuice transfection of COS-7 and HeLa cells with rhodamine-labeled DNA

6
Cells grown on polylysine-coated coverslips to 50% confluency were transfected with rhodamine-labeled pTriEx™-2 DNA using GeneJuice. Labeled
plasmid DNA was complexed with GeneJuice in serum-free medium, and the complexes were added directly to the cells in complete medium. Twenty-
four hours after transfection, the cells were washed in PBS, fixed in 4% formalin for 10 minutes, and washed again in PBS. Coverslips were mount-
ed on glass slides and sealed for confocal microscopy. The transfected DNA is seen in red. Unfiltered reflected light from the 533 laser was collect-
ed to image the cell boundaries.
7
Antibiotics Product Size Cat. No.
Reliable selection reagents for mammalian cell culture G 418 Sulfate 100 mg 345810
Cell Culture-Tested 250 mg
G 418 Sulfate causes misreading of the mRNA. The hph gene
500 mg
1g 8
encodes resistance to Hygromycin B. 5g
G 418 sulfate is an aminoglycoside that
Cat. No. 400052 is a sterile aqueous solution G 418 Sulfate, 10 ml 345812
inhibits prokaryotic and eukaryotic protein syn-
at 50 mg/ml in PBS. Purity: ≥ 90% by HPLC and Sterile-Filtered 20 ml
thesis. Widely used in the selection of eukaryotic
TLC. Potency: ≥ 1000 µg/mg solid. Aqueous Solution, 50 ml
vectors carrying neomycin or kanamycin resis- Cell Culture-Tested
tance genes. The product of these genes, amino-
glycoside 3'-phosphotransferase, inactivates
Cat. No. 400053 is a a sterile aqueous solu-
tion at 50 mg/ml in 25 mM HEPES, pH 7.2. Hygromycin B, 5 ml 400052 9
Purity: ≥ 90% by HPLC and TLC. Potency: ≥ 1000 Streptomyces sp., 20 ml
G 418, neomycin, and kanamycin by phosphoryla- Sterile-Filtered Solution 50 ml
µg/mg solid. in PBS, Cell Culture-Tested
tion. Purity: ≥ 98% by TLC. Potency: ≥ 730 µg/mg.
RTECS WK2130000, CAS 31282-04-9, M.W.
Cat. No. 345810 is a white solid. Soluble in Hygromycin B, 5 ml 400053
527.5
aqueous buffers and water. Cat. No. 345812 is a Streptomyces sp., 20 ml
liquid formulation of Cat. No. 345810, sterile-fil-
Risk and Safety Statements:
Cat. No. 400052 R: 26/27/28-37/38-41; S: 23-26-36/37/39-45
Sterile-Filtered Solution
in 25 mM HEPES, Cell Culture-Tested
10
tered at 50 mg/ml active antibiotic.
Cat. No. 400053 R: 26/27/28-37/38-41; S: 22-26-36/37/39-45
RTECS CB9378500, CAS 108321-42-2, M.W. Neomycin Sulfate 10 g 4801
692.7 25 g
Neomycin Sulfate
Risk and Safety Statements: Neomycin Sulfate,
g-Irradiated, Tissue
Cat. Nos. 345810, 345812 R: 61; S: 36/37/39-45-53 Neomycin sulfate is an aminoglycoside
antibiotic that inhibits translation by binding to Culture Grade 20 ml 480100 11
Hygromycin B the small subunit of prokaryotic ribosomes.
Appearance: off-white solid. Soluble in water.
Hygromycin is a unique aminoglycoside antibi-
RTECS QP4375000, CAS 1405-10-3, M.W. 908.9
Appendices
otic that inhibits the growth of prokaryotic (bacte-

Risk and Safety Statements:
Indices
ria) and eukaryotic microorganisms (yeasts) and

Cat. No. 4801 R: 36/37/38-42/43-63; S: 26-36/37/39-45
mammalian cells. It inhibits protein synthesis at
Cat. No. 480100 R: 36/37/38-42/43-63; S: 26-36/37/39-45
the translocation step on the 70S ribosome and

131
Novagen
Protein Expression
Multisystem Expression
1 pTriEx™ Multisystem Expression Vectors Product Size Cat. No.
One construct for efficient expression in bacterial, insect and mammalian systems pTriEx™-1.1 DNA 20 µg 70840-3
Target genes are often expressed in more • Infection-induced expression in insect cells
than one system for various purposes. For exam- from the AcNPV baculovirus p10 promoter
ple, a bacterial system may be used for initial • Transient expression in vertebrate cells from
2 studies to ascertain solubility or activity, or to
produce large amounts of protein for structural
a choice of b-actin (CAG) promoter or CMV
promoter
TriExUP Primer 500 pmol 70846-3
TriExDOWN Primer 500 pmol 70847-3
studies or antibody production. The same gene
• RNA generated from CAG or CMV promoter Product Cat. No.
may need to be expressed in insect and/or mam-
possesses an intron designed to facilitate
malian cells, in order to obtain higher activity or pTriEx-1.1 Cloning Kit 70898-3
mRNA processing and export
eukaryotic post-translational modifications. pTriEx-2 Cloning Kit 70866-3
3 Although Novagen’s bacterial and baculovirus
• Unique 3-ORF MCS enables in-frame cloning
of any blunt insert pTriEx-3 Cloning Kit 70873-3
expression vectors offer compatible cloning
strategies and restriction sites to make the • N-terminal His•Tag® sequence for easy pTriEx-4 Cloning Kit 70879-3
cloning process more convenient, the entire pro- detection and purification (pTriEx-2 and -4)
Components:
cess can be streamlined by using a single vector • Optional C-terminal HSV•Tag® and His•Tag • 20 µg pTriEx Vector
4 to reliably express target genes in the three

major expression systems. The benefits of such •
sequences
N-terminal S•Tag™ sequence for sensitive
• 55 µl
• 10 µl
Clonables™ 2X Ligation Premix
Clonables Positive Control
a multisystem vector are enhanced when per- homogeneous FRET assay of fusion proteins • 1.5 ml Nuclease-free Water
forming high throughput (HT) gene analysis, (pTriEx-2 and -4) • 11 × 50 µl NovaBlue Singles™ Competent
which currently requires a significant effort to Cells
• Multisystem transcription
construct and manage all of the multiple recom- • 2 × 2 ml SOC Medium
terminators/polyadenylation site
5 binants used for different expression systems.
To address this need, Novagen has devel-
• pTriEx-4 available as a LIC Vector for rapid
• 20 µl
Product
Test Plasmid
Cat. No.
directional cloning of PCR products
oped the pTriEx™ System, a novel expression
• Ideally positioned protease sites for cleavage TriEx Bacterial
vector platform that enables optimal protein Expression System 1.1 70900-3
expression in bacterial, insect and mammalian of N-terminal tags to generate native proteins
• High-copy origin of replication TriEx Bacterial
cells from a single plasmid.
6 Features The pTriEx system thus combines the proven
Expression System 2
TriEx Bacterial
70867-3
• A family of multisystem expression vectors to features of the pET, BacVector® and mammalian Expression System 3 70892-3
minimize DNA cloning manipulations systems and provides all of the benefits and ver-
satility of each expression system in one vector TriEx Bacterial
• Optimized transcription and translation Expression System 4 70895-3
backbone. The pTriEx vectors have been exten-
signals for E. coli, baculovirus and
7 mammalian expression
sively tested for performance with various target
genes in all three expression systems. In each
Components:
• 20 µg pTriEx Vector
• IPTG-inducible expression in E. coli from the case, expression was similar to that obtained • 0.2 ml Tuner(DE3)pLacI Competent Cells
tightly controlled T7lac promoter • 0.2 ml Origami(DE3)pLacI Competent
with system-specific vectors (see next page).
Cells
• 2 × 2 ml SOC Medium
8 pTriEx-1.1 pTriEx-2
• 20 µl Test Plasmid
product listing continued on next page

CMV ie promoter pTriEx-3 pTriEx-4
T7lac T7lac
CMV ie enhancer Chicken ß-actin promoter
p10 p10
9 6 03 Exon 1
intron
Nco I
EcoR V
Sma I
Nco I
His•Tag
Xcm I
2
Ecl136 I S•Tag
le f-
BamH I thrombin site

E xon 2
EcoR I Sma I
Bgl II enterokinase site
Asc I PshA I
BssH I* BseR I
Rabbit ß-globin terminator
Pst I EcoR V
10 T7 terminator
Sse8387 I Ecl136 I
Ap
Kpn I BamH I
PinA I EcoR I
29
16 Nsp V Bgl II
Hind III Asc I
o ri Not I BssH I*
Eag I* Pst I
Pvu II Sse8387 I
actin promoter CMV promoter Bst1107 I Kpn I
Pml I PinA I
11 pTriEx-1.1
pTriEx-2
pTriEx-3
pTriEx-4 HSV•Tag
Xho I
His•Tag
Nsp V
Hind III
Not I
Dra III Eag I*
Bsu36 I Pvu II
Bst1107 I
* not unique in pTriEx-1.1 or pTriEx-2 Pml I
Appendices
HSV•Tag
Xho I
Indices
His•Tag
Dra III
Bsu36 I

Novagen
Protein Expression
Multisystem Expression
pTriEx™ Multisystem Expression Vectors continued

1
1 2 3 4 5 6 7 8 9 M kDa Available separately:
– 225 1. uninduced pTriEx-1
– 150 2. pTriEx-1.1 EGFP (27 kDa)
– 100 3. pET-28b(+) EGFP (27 kDa) Clonables™ Ligation/
– 75 4.
5.
pTriEx-1.1 LUC (61 kDa)
pET-28b(+) LUC (61 kDa)
Transformation Kit 11 rxn 70526-3 2
– 50 NovaBlue 0.4 ml 69825-3
6. pTriEx-1.1 b-gal (119 kDa)
– 35 Competent Cells 1 ml 69825-4
7. pET-28b(+) b-gal (119 kDa)
– 25 Tuner™(DE3)pLacI 0.4 ml 70625-3
8. pTriEx-1.1 GUS (72 kDa)
9. pET-28b(+) GUS (70 kDa) Competent Cells 1 ml 70625-4
– 15 M. Perfect Protein™ Markers
Origami™(DE3)pLacI
Competent Cells
0.4 ml
1 ml
70629-3
70629-4
3
Origami B(DE3)pLacI 0.4 ml 70838-3
E. coli expression of 4 target proteins from pTriEx-1.1 and pET-28b(+) Competent Cells 1 ml 70838-4
The indicated target genes were cloned into pTriEx-1.1 and pET-28b(+) using Nco I as the amino terminal cloning site. For protein expression,
pTriEx-1 recombinants were grown in host BL21(DE3)pLacI and pET-28b(+) recombinants were grown in host BL21(DE3). Cells were induced at Rosetta™(DE3)pLacI 0.4 ml 70920-3
mid-log phase by the addition of 1 mM IPTG and cultured for an additional 3 h at 37°C. Harvested cell pellets were resuspended in 1X SDS
sample buffer and equivalent amounts of total cell protein loaded on a 4–20% gradient gel. The gel was stained with Coomassie blue. Rosetta-gami™(DE3)pLacI 4
Competent Cells 0.4 ml 71056-3
1 ml 71056-4
BacVector®-3000
Transfection Kit 12 rxn 70077-3
5
M 1 2 3 4 5 6 M kDa
1. pBAC-2cp GUS (70 kDa) GeneJuice™ 1 ml 70967-3
– 150
2. pTri-Ex-1.1 GUS (72 kDa) Transfection Reagent 10 ml 70967-4
– 100 0.3 ml 70967-5
– 75 3. pBAC-2cp EGFP (27 kDa)
4. pTriEx-1.1 EGFP (27 kDa)
– 50 5. pBAC-2cp β-gal (119 kDa)
– 35 6. pTriEx-1.1 β-gal (119 kDa) Additional Information Available
– 25 M. Perfect Protein Markers TriEx System Manual
TB250
www.novagen.com 6
– 15
Vector Cloning Region Sequence Appendix
inNovations No. 10
Baculovirus expression of 3 target proteins from pTriEx-1.1 and pBAC-2cp recombinants

The indicated target genes were cloned into pTriEx-1.1 and pBAC-2cp using Nco I as the amino terminal cloning site, and baculovirus recombi-
nants were constructed using BacVector®-3000 Triple Cut Virus DNA. For protein expression, titered stocks of the recombinant baculoviruses 7
were used to infect Sf9 cells grown in shaker culture in BacVector Insect Cell Medium at MOI = 5. Harvested cells were resuspended in PBS.
After addition of an equal volume of 2X SDS sample buffer, total cell protein samples corresponding to 2 × 104 cells were loaded on 4–20% gra-
dient gels. The gels were stained with Coomassie blue.
8
Luciferase Assay GUS Assay
3
500
nmoles MU released/min per 106 cells
Relative Luminescence
9
Units per 104 cells
400
2
300
200
1
100
0
10
cells 0
pBacMam-2 pTriEx-1 alone vTriEx-GUS vBacMam-3 GUS Cells
pBacMam-3 pcDNA3.1 Virus
Mammalian expression of 2 target proteins from pTriEx-1.1 and pBacMam recombinants

The indicated target genes were cloned into pTriEx-1.1, pBacMam plasmids and pcDNA3.1 (Invitrogen). The LUC recombinants were transfected
into COS-7 cells and LUC activity measured in cell lysates after 24 h (left panel). The GUS recombinants were used to construct recombinant
11
baculoviruses using BacVector-3000 Triple Cut Virus DNA, which were used to transduce COS-7 cells at 100–200 pfu per cell. After 24 h, GUS
activity was measured in cell lysates (right panel).
Appendices
Indices

133

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Novagen

Novagen Tools in this Chapter

Insect Cell Expression

Multisystem Expression Fusion Tag Assay

82 Novagen 2002–2003 Catalog

Novagen 2002–2003 Catalog 83

1 pETBlue™ and pET System Overview

3 sion occurs until a source of T7 RNA polymerase

7 nology are represented by the pETBlue™ System

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pETBlue™ and pET System Overview continued

Fusion Tags Protease

pET-40b(+) T7lac Kan DsbA•Tag/His•Tag/S•Tag His•Tag SP, Tb, Ek periplasm.

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1 pETBlue™ System Product Cat. No.

Vector Insert Vector Insert

9 TTCCTCTATATCTA TAC XXX

8 bp spacing between RBS and ATG

6 bp spacing between RBS and ATG

pETBlue-1: Two alternative designs of insert 5'-ends for optimal expression

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pETBlue™ PCR Cloning Kits pETBlue™ AccepTor™ Vector Kits

pETBlue-1 Perfectly Blunt

pETBlue-1 and pETBlue-2 Blunt Vectors

AccepTor Vector Kits Protocol TB248

pETBlue System Protocol TB249

HSV•Tag His•Tag Vector Cloning Region Sequences Appendix

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1 pET System Tutorial

T7 gene 1 Target gene

the system in greater detail.

7 scription in the uninduced state and in the

9 and, therefore, represent the most stringent con-

10 used hosts are BL21 and its derivatives, which

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pET System Tutorial continued pET System Advantages

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1 pET System Tutorial continued

Choosing a pET Vector lowing purification by digestion with a site-spe-

combination of vector, host strain, and culture

sion, folding can be improved in hosts that are

these cases, an alternative is to construct a

fusion protein with efficiently expressed amino

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pET System Tutorial continued

sequences without the additional DsbA or DsbC pET-30a–c(+) pET-40b(+)

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1 pET System Tutorial continued

2 single preparation of insert can be used with all

Biol. 189, 113–130.

5 3. Studier, F. W., Rosenberg, A. H., Dunn, J. J., and

J. (1993) Science 262, 1744–1747.

7 (2001) inNovations 12, 1-3.

inNovations 1, 1–3. periplasm/media covalent immobilization

8 Dsb•Tag™ N 208 (DsbA) potential periplasmic

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pET Vector DNA 1

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1 pET Expression Systems