Novagen

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Protein Expression
Prokaryotic Expression

pET System Tutorial

The premier E. coli expression system
The pET System is the most powerful system
yet developed for the cloning and expression of
recombinant proteins in E. coli. Based on the T7
promoter-driven system originally developed by
Studier and colleagues (13), Novagens pET
System has been used to express thousands of
different proteins. Please refer to pET System
Overview earlier in this chapter for the basic
features of the pET System. This section
describes some of the unique characteristics of
the system in greater detail.

Figure 1. Control elements of the pET System

IPTG Induction

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Appendices
Indices

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88

Thirteen different host strains are available

as lDE3 lysogens (see Table 1). The most widely
used hosts are BL21 and its derivatives, which
have the advantage of being deficient in both lon
(5) and ompT proteases. The B834 strain is a
methionine auxotroph and, therefore, enables
high specific activity labeling of target proteins
with 35S-methionine or selenomethionine (6).
The BLR strain is a recA derivative that
improves plasmid monomer yields and may help
stabilize target plasmids containing repetitive
sequences. Two thioredoxin reductase (trxB)
mutant strains (AD494, BL21trxB) are available
that facilitate disulfide bond formation in the E.
coli cytoplasm (7). The Origami, Origami B
continued on next page

INACTIVE

lac
repressor

lac
repressor

lac I gene

pET

lac I gene
T7 lysosyme
pLysS
or E
T7 lysozyme
gene

HOST CELL

E. coli genome

Table 1. pET System Host Strains

Antibiotic Resistance
Kan
Kan
Kan + Cam
none
none
Cam
Cam
Kan
Kan + Cam
Tet
Tet
Tet + Cam

Available as
Competent
Cells
yes
yes
yes
yes
yes
yes
no
yes
yes
yes
yes
yes

met auxotroph; 35S-met and

selenomethionine labeling

none
none
Cam

no
yes
yes

K-12

recA mutant
Rif resistance

Rif
Rif
Rif + Cam
Rif + Cam

yes
yes
yes
no

NovaBlue
NovaBlue(DE3)

K-12

recA, endA, lacIq

cloning, plasmid preps

Tet
Tet

yes
yes

Origami
Origami(DE3)
Origami B(DE3)pLysS

K-12

trxB/gor mutant, greatly facilitates

cytoplasmic disulfide bond formation

Kan + Tet
Kan + Tet
Kan + Tet + Cam

yes
yes
yes

Origami B
Origami(DE3)
Origami B(DE3)pLysS

Tuner

Rosetta
Rosetta(DE3)
Rosetta(DE3)pLysS
Rosetta-gami
Rosetta-gami(DE3)
Rosetta-gami(DE3)pLysS

Tuner

BL21 lacY deletion, trxB/gor mutant,

greatly facilitates cytoplasmic disulfide
bond formation; allows precise control
with IPTG
Enhances expression of
proteins having codons rarely used in
E. coli, lacY deletion mutant
Enhances expression of
proteins having codons rarely used in
E. coli, trxB/gor mutant

Kan + Tet
Kan + Tet
Kan + Tet + Cam
Kan + Tet + Cam
Cam
Cam
Cam
Kan + Tet + Cam
Kan + Tet + Cam
Kan + Tet + Cam

yes
yes
yes
yes
yes
yes
yes
yes
yes
yes

RosettaBlue
RosettaBlue(DE3)
RosettaBlue(DE3)pLysS

NovaBlue

Enhances expression of
proteins having codons rarely used in
E. coli, recA, endA, lacIq

Tet + Cam
Tet + Cam
Tet + Cam

yes
yes
yes

Tuner
Tuner(DE3)
Tuner(DE3)pLysS

BL21

BL21 lacY deletion mutant

allows precise control with IPTG

none
none
Cam

yes
yes
yes

Strain
AD494
AD494(DE3)
AD494(DE3)pLysS
BL21
BL21(DE3)
BL21(DE3)pLysS
BL21(DE3)pLysE
BL21trxB(DE3)
BL21trxB(DE3)pLysS
BLR
BLR(DE3)
BLR(DE3)pLysS

Derivation
K-12

Key Feature(s)
trxB mutant; facilitates cytoplasmic
disulfide bond formation

B834

Lacks lon and ompT

proteases

BL21

BL21 trxB mutant; facilitates

cytoplasmic disulfide bond formation
BL21 recA mutant; stabilizes tandem
repeats

B834
B834(DE3)
B834(DE3)pLysS

B strain

HMS174
HMS174(DE3)
HMS174(DE3)pLysS
HMS174(DE3)pLysE

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lac o
T7 promoter

DE3

Host Strains
After plasmids are established in a nonexpression host, they are most often transformed into a host bearing the T7 RNA polymerase gene (lDE3 lysogen) for expression of
target proteins. Figure 1 illustrates in schematic
form the host and vector elements available for
control of T7 RNA polymerase levels and the
subsequent transcription of a target gene in a
pET vector. In lDE3 lysogens, the T7 RNA polymerase gene is under the control of the lacUV5
promoter. This allows some degree of transcription in the uninduced state and in the
absence of further controls is suitable for
expression of many genes whose products have
innocuous effects on host cell growth. For more
stringent control, hosts carrying either pLysS or
pLysE are available. The pLys plasmids encode
T7 lysozyme, which is a natural inhibitor of T7
RNA polymerase, and thus reduces its ability to
transcribe target genes in uninduced cells. pLysS
hosts produce low amounts of T7 lysozyme,
while pLysE hosts produce much more enzyme
and, therefore, represent the most stringent control available in lDE3 lysogens (4).

Target gene

T7 RNA polymerase

lac o
lac promoter

The pET System provides six possible

vector-host combinations that enable tuning of
basal expression levels to optimize target gene
expression (2). These options are necessary
because no single strategy or condition is suitable for every target protein.

Thirteen different host strains

10

T7 RNA
polymerase
T7 gene 1

Control Over Basal Expression Levels

IPTG Induction

E. coli RNA
polymerase

BL21

Origami

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Protein Expression
Prokaryotic Expression

pET System Tutorial

pET System Advantages

continued

Powerful
Promoter
Host BL21(DE3)

Recombinant antibody
T7lac
T7
T7lac T7lac

pLysE
pLysS
(uninduced)

T7lac
pLysE

T7

Human p53
T7
T7lac
pLysS

Highest expression levels, tightest control

over basal expression

Choice of vectors and host strains to

control basal and induced expression levels

Precise control of induced expression with

IPTG in Tuner and Rosetta hosts

75

Rare codons supplied by Rosetta hosts

50

Origami strains for enhanced disulfide

bond formation in the cytoplasm

T7lac
pLysS
kDa
150
100

p53

35
rec. ab
25

The indicated cell cultures were grown at 37C to OD600 of approximately 0.8 and expression induced with 1 mM IPTG for 2.5 h. Total
cell protein samples were run along with Novagens Perfect Protein Markers on a 420% 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.

and Rosetta-gami strains are double mutants

of trxB/gor, which are the key enzymes in both
major reductive pathways (8). These hosts thus
represent a significant advantage for the formation of properly folded disulfide-containing proteins. The Rosetta, Rosetta-gami, and
RosettaBlue strains supply the tRNAs for six
codons used only rarely in E. coli, which alleviates poor expression caused by incompatible
codon usage in some eukaryotic genes (9). The
K-12 derivative strains HMS174, NovaBlue, and
RosettaBlue are recA, like BLR. These strains
may stabilize certain target genes whose products may cause the loss of the DE3 prophage.
NovaBlue and RosettaBlue are potentially useful
as stringent hosts due to the presence of the high
affinity lacIq repressor encoded by the F episome. In addition, Novagen offers the lDE3
Lysogenization Kit for making new expression
hosts with other genetic backgrounds. An alternative for expressing extremely toxic genes or
preparing a new lDE3 lysogen is to provide T7
RNA polymerase by infection with lCE6.
Although not as convenient as inducing a lDE3
lysogen with IPTG, this strategy may be preferred
for certain applications.
High Stringency T7lac Promoter

Choice of N-terminal and C-terminal fusion

tags for detection, purification and
localization

Expanded multiple cloning sites in all three

reading frames

f1 origin of replication for mutagenesis and

sequencing

E. coli-based system for rapid results

Convenient restriction sites for subcloning

from other vectors

Choice of methods for one-step purification

of target proteins without antibodies

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Complete

Variety of system configurations plus many

supporting products

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Control Over Induced Expression Levels

In many cases the expression of optimal

levels of active, soluble protein depends on
host cell background, culture conditions, and
vector configuration; often the conditions for
highest activity of a target protein do not correlate with conditions that produce the highest mass of target protein. In addition to
offering variable stringency based on
vector/host combinations that provide control
over basal expression of T7 RNA polymerase,
the pET System offers precise control over
target protein expression based on inducer
(IPTG) concentration, made possible by the
lacY mutation in the Tuner, Rosetta, and
Origami B host strains.

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continued on next page

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Appendices
Indices

In addition to the choice of three basic

expression stringencies at the host level, the
pET system provides two different stringency
options at the level of the T7 promoter itself: the
plain T7 promoter and the T7lac promoter (8;
also shown in Figure 1). The T7lac promoter
contains a 25 bp lac operator sequence imme-

diately downstream from the 17 bp promoter

region. Binding of the lac repressor at this site
effectively reduces transcription by T7 RNA
polymerase, thus providing a second lacI-based
mechanism (besides the repression at lacUV5) to
suppress basal expression in lDE3 lysogens.
pET plasmids with the T7lac promoter also carry
their own copy of lacI to ensure that enough
repressor is made to titrate all available operator
sites.
In practice, it is usually worthwhile to test
several different vector/host combinations to
obtain the best possible yield of protein in its
desired form (10). Figure 2 illustrates dramatic
differences in the expression of two target proteins with various combinations.

Versatile

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Figure 2. Effect of vector/host combination on expression levels of two proteins

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Prokaryotic Expression
continued

Choosing a pET Vector

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4

A wide variety of pET vectors is available. All

are derived from pBR322 and vary in leader
sequences, expression signals, fusion tags, relevant restriction sites, and other features. There
are two major categories of pET plasmids
known as transcription vectors and translation
vectors:
The transcription vectors [including pET21(+), pET-23(+), and pET-24(+)] express
target RNA but do not provide translation
signals. They are useful for expressing
proteins from target genes that carry their
own bacterial translation signals. Note that
the transcription vectors can be identified
by lack of a letter suffix after the name.

The translation vectors contain efficient

translation initiation signals and are designed
for protein expression. Most contain cloning
sites in reading frames a, b, or c that
correspond to the GGA, GAT, or ATC triplet
of the BamH I site, respectively.

Primary Considerations

Appendices
Indices

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90

Cloning strategy

Applications for proteins expressed in pET

vectors vary widely. For example, analytical
amounts of a target protein may be needed for
activity studies, screening and characterizing
mutants, screening for ligand interactions, and
antigen preparation. Large amounts of active
protein may be required for structural studies,
use as a reagent, or affinity matrix preparation.
Any number of vectors may be suitable for
expression of analytical amounts of protein for
screening or antigen preparation, yet only one
combination of vector, host strain, and culture
conditions may work best for large scale purification. If a high yield of active protein is needed
on a continual basis, it is worth testing a matrix
of vector, host, and culture combinations to find
the optimal result.
Any information available about the target
protein may help determine the choice of vector.
For example, some proteins require no extraneous sequence on one or both termini for activity.
Most pET vectors enable cloning of unfused
sequences; however, expression levels may be
affected if a particular translation initiation
sequence is not efficiently utilized in E. coli. In
these cases, an alternative is to construct a
fusion protein with efficiently expressed amino
terminal sequences (available with many pET
vectors) and then remove the fusion partner fol-

T7

an

Solubility and Cellular Localization

Once you have considered your application
and cloning strategy, a good starting point for
any expression project is to determine the cellular localization and solubility of the target protein. In many applications, it is desirable to
express proteins in their soluble, active form.
Solubility of a particular target protein is
determined by a variety of factors, including the
individual protein sequence. In most cases, solubility is not an all-or-none phenomenon; the vector, host, and culture conditions can be used to
increase or decrease the proportion of soluble
and insoluble forms obtained. The choice of vector and expression host can significantly
increase the activity and amount of target protein present in the soluble fraction. A vector can
enhance solubility and/or folding in three ways:
1, provide for fusion to a polypeptide that itself
is highly soluble (e.g., GST, thioredoxin, NusA),
2, provide for fusion to an enzyme that catalyzes
disulfide bond formation (e.g., thioredoxin,
DsbA, DsbC), or 3, provide a signal sequence for
translocation into the periplasmic space. When
using vectors designed for cytoplasmic expression, folding can be improved in hosts that are
permissive for the formation of disulfide bonds
in the cytoplasm. The thioredoxin reductase
(trxB) mutation has been shown to allow the
formation of disulfide bonds in the E. coli cytoplasm, which is further enhanced by the additional mutation in the glutathione reductase
(gor) gene in Origami and Rosetta-gami
hosts (8). Induction at lower temperatures
(1525C) can also increase the proportion of
soluble target proteins.
The pET-43.1 and pET-32 vectors incorporate
fusion tags specifically designed to enhance the
solubility of target proteins in the E. coli

pET-9ad

ri

f1 origin

pET-20b(+)
pET-23(+)
pET-23ad(+)

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pET vector backbones: plain T7 promoter vectors

The pET vectors shown here and on the facing page are grouped
according to the functional elements present on the plasmid backbones. Features of the T7 cloning and expression regions (indicated
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
pBR322 convention, so that the T7 expression region is reversed on
these maps and in the published sequences.

continued on next page

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Specific information known about the

expressed protein

pET-3ad
pET-12ac
pET-14b
pET-17b
pET-17xb

T7

Choosing a pET vector for expression usually involves a combination of factors. Consider
the following three primary factors:
The application intended for the expressed
protein

lowing purification by digestion with a site-specific protease. The LIC (ligation-independent

cloning) strategy is especially useful for this
approach, because the cloning procedure
enables the removal of all amino terminal vectorencoded sequences with either enterokinase or
Factor Xa .
Cloning strategies can affect the choice of
vector due to the need for restriction site and
reading frame compatibilities. Because many of
the pET vectors share common restriction site
configurations, it is usually possible to clone a
target gene into several vectors with a single
preparation of the insert. Different considerations apply when using PCR cloning strategies.
The LIC Vector Kits are recommended for this
purpose, and enable the preparation of inserts
by PCR and eliminate the need for restriction
digestion of vector or insert. The Appendix contains sequences of cloning and expression
regions for the pET vectors.

pET System Tutorial

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Protein Expression

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Protein Expression
Prokaryotic Expression
continued

Fusion Tags for Different Needs

la

2
la cI

pET-11ad
pET-15b
pET-16b
pET-19b

T7
la

f1 origin

lac I

pET-21(+)
pET-21ad(+)
pET-22b(+)
pET-25b(+)
pET-31b(+)
pET-32ac(+)
pET-32 Ek/LIC
pET-32 Xa/LIC
pET-43.1ac(+)*

ri
* Ap gene in opposite orientation in
pET-43.1 series

f1 origin

T7
l

pET-24(+)
pET-24ad(+)
pET-26b(+)
pET-27b(+)
pET-28ac(+)
pET-29ac(+)
pET-30ac(+)
pET-30 LIC
pET-33b(+)

pET-34b(+)
pET-35b(+)
pET-36b(+)
pET-37b(+)
pET-38b(+)
pET-39b(+)
pET-40b(+)
pET-41ac(+)
pET-42ac(+)

l acI

an

ri

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pET vector backbones:
T7lac promoter vectors

11
Appendices
Indices

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continued on next page

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T7

If a fusion sequence is tolerated by the application you are using, it is useful to produce
fusion proteins carrying the STag, T7Tag,
GSTTag, HisTag, or HSVTag peptides for
easy detection on Western blots. These peptides
are small in size (except for the GSTTag
sequence) and the detection reagents for them
are extremely specific and sensitive. The
HisTag, GSTTag, STag, and T7Tag
sequences can also be used for affinity purification using the corresponding resins and buffer
kits.
Fusion proteins can be accurately quantified
in crude extracts or purified form using STag
and GSTTag Assay Kits. The FRETWorks
STag Assay Kit is based on a novel substate
that enables fluorescent detection of less than
1 fmol of fusion protein in a homogeneous format. (See Chapter 7, Protein & Gene Analysis.)
The HisTag sequence is very useful as a
fusion partner for purification of proteins in general. It is especially useful for those proteins initially expressed as inclusion bodies, because
affinity purification can be accomplished under
totally denaturing conditions that solubilize the
protein.
The CBDTag sequences are also generally
useful for low cost affinity purification. They are
also uniquely suited to refolding protocols [especially pET-34b(+) and 35b(+), which contain the
CBDclosTag sequence]. Because only properly
refolded CBDs bind to the cellulose matrix, the
CBIND affinity purification step can remove
improperly folded molecules from the preparation. While any of the tags can be used to immobilize target proteins, the CBDTag sequences
are ideally suited for this purpose due to the
inherent low non-specific binding and biocompatibility of the cellulose matrix.
The NusTag, TrxTag and GSTTag
sequences have been reported to enhance the
solubility of their fusion partners. The NusTag
and TrxTag vectors are compatible with
Origami, Origami B, and Rosetta-gami host
strains, which facilitate disulfide bond formation
in the cytoplasm.
The various fusion tags available and their
corresponding pET vectors are listed in the following table. A number of pET vectors encode
several of the fusion tags in tandem as aminoterminal fusion partners. In addition, many vectors enable expression of fusion proteins carrying a peptide tag on each end by allowing inframe read-through of the target gene sequence.
Using vectors with protease cleavage sites
(thrombin, Factor Xa, enterokinase) between the
amino terminal tag and the target sequence
enables optional removal of one or more tags
following purification. Vectors that represent a

cytoplasm. These vectors are also compatible

with trxB mutant hosts AD494 and BL21trxB,
and with the trxB/gor mutant Origami,
Origami B, and Rosetta-gami strains. The pET43.1ac(+) vectors incorporate the 495 aa solubility-promoting NusA (NusTag) sequence,
which was discovered through a systematic
search for E. coli proteins that have the highest
potential for solubility when overexpressed (11).
Many proteins that are normally produced in an
insoluble form in E. coli tend to become more
soluble when fused with the 109 aa N-terminal
thioredoxin (TrxTag) sequence. The TrxTag
expressed from pET-32 vectors not only
enhances the solubility of many target proteins,
but appears to catalyze the formation of disulfide bonds in the cytoplasm of trxB mutants
(12).
Schistosomal glutathione-S-transferase (GST)
is commonly used as an N-terminal fusion partner when expressing proteins in E. coli.
Although not specifically designed for this purpose, the 220 aa GSTTag sequence has been
reported to enhance the solubility of its fusion
partners. The pET-41 and -42 series of vectors
encode the GSTTag sequence driven by the
powerful T7lac promoter. Note that these vectors carry kanamycin resistance so are not recommended for use with trxB mutant hosts.
An alternative strategy to obtain active, soluble proteins is to use vectors that enable export
into the periplasm, which is a more favorable
environment for folding and disulfide bond formation. For this purpose vectors carrying signal
peptides are used. DsbA and DsbC are periplasmic enzymes that catalyze the formation and isomerization of disulfide bonds, respectively. The
208 aa DsbATag [pET-39b(+)] and 236 aa
DsbCTag [pET-40b(+)] vectors enable fusion
of target polypeptides to these enzymes, which
include their N-terminal secretion signals. If the
fusion protein is exported to the periplasm, the
Dsb partner can assist in proper disulfide bond
formation. Other pET vectors that carry signal
sequences without the additional DsbA or DsbC
coding regions are also available.
Some purification strategies optimize production of insoluble inclusion bodies in the cytoplasm. Inclusion bodies are extracted and solubilized; then the target protein is refolded in vitro,
(e.g., with Novagens Protein Refolding Kit). This
procedure usually produces the highest yields of
initial protein mass and protects against proteolytic degradation in the host cell. However, the
efficiency of refolding into active protein varies
significantly with the individual protein and can
be quite low. The pET-31b(+) vector is specifically designed for the generation of insoluble
fusion proteins and provides a powerful method
for the production of small proteins and peptides.

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Protein Expression
Prokaryotic Expression

pET System Tutorial

continued

good selection for cellular localization and affinity tag configurations are pET-30 Ek/LIC, pET-32
Ek/LIC, pET-41 Ek/LIC and pET-43.1 Ek/LIC. A
single preparation of insert can be used with all
of the ready-to-use Ek/LIC vectors to allow convenient construction of several target gene configurations at once.
How to Order

3
4

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6
7

The pET Vectors are available configured

into Expression Systems containing one or more
vectors, plus glycerol stocks and competent
cells of popular host strains for cloning and
expression. Individual plasmids and cell strains
are available separately.
1.

Moffatt, B. A., and Studier, F. W. (1986) J. Mol.

Biol. 189, 113130.

2.

Rosenberg, A. H., Lade, B. N., Chui, D., Lin, S.,

Dunn, J. J., and Studier, F. W. (1987) Gene 56,
125135.

3.

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

Dubendorff, J. W. (1990) Methods Enzymol. 185,
6089.

4.

Studier, F. W. (1991) J. Mol. Biol. 219, 3744.

5.

Phillips, T. A., Van Bogelen, R. A., and Neidhardt, F.

C. (1984) J. Bacteriol. 159, 283287.

6.

Leahy et al. (1992) Science 258, 987991.

7.

Derman, A. I., Prinz, W. A., Belin, D., and Beckwith,

J. (1993) Science 262, 17441747.

8.

Dubendorff, J. W. and Studier, F. W. (1991) J. Mol.

Biol. 219, 4559.

9.

Novy, R., Drott, D. Yaeger, K., and Mierendorf, R.

(2001) inNovations 12, 1-3.

Table 2. Fusion tags available for pET constructs

Tag

N/C
Terminal
or Internal

Size (aa)

Basis for Detection

and/or Purification

Applications

pET Vector Series

T7Tag

N, I

11 or 260

monoclonal antibody

Western blot,
immunoprecipitation,
purification

3, 9, 11, 17 17x,
21, 23, 24, 28, 33

STag

N, I

15

S-protein (104 aa) affinity

Western blot,
quantitative assay,
purification

29, 30, 32, 3437

39, 40, 41, 42,
43.1

HisTag

N, C, I

6, 8, or 10

metal chelation
chromatography
(native or denaturing)

HisBind resin
purification

14-16, 1943.1

HSVTag

11

monoclonal antibody

Western blot,
immunofluorescence

25, 27, 43.1

pelB/ompT

20/22

potential periplasmic
localization

protein export/folding

12, 20, 22
25, 26, 27

KSI

125

highly expressed
hydrophobic domain

small protein/peptide
production/purification,
insoluble protein

31

TrxTag

109

thioredoxin

soluble protein, cytoplasmic disulfide bond

formation in trxB hosts

32

PKA site

protein kinase A
recognition site

in vitro phosphorylation

33

CBDclosTag

156

polyclonal antibody,
cellulose binding
domain

Western blot,
purification, noncovalent
immobilization

34, 35

CBDcenATag

114

polyclonal antibody,
cellulose binding domain,
periplasm/media

protein export, Western

blot, purification, noncovalent immobilization

36, 37

CBDcexTag

107

polyclonal antibody,
cellulose binding domain,
periplasm/media

protein export, Western

blot, purification, noncovalent immobilization

38

DsbTag

208 (DsbA) potential periplasmic

236 (DsbC) localization

soluble protein, periplasmic disulfide bond

formation, isomerization

39, 40

GSTTag

220

glutathione affinity
monoclonal antibody
enzymatic activity

purification,
Western blot,
quantitative assay

41, 42

NusTag

495

NusA

soluble protein, cytoplasmic disulfide bond

formation in trxB hosts

43.1

10. Mierendorf, R., Yaeger, K., and Novy, R. (1994)

inNovations 1, 13.
11. Harrison, R. (2000) inNovations 11, 47
12. Stewart et al. (1998) EMBO J. 17, 55435550.

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9
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Appendices
Indices

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