CHAPTER SEVENTEEN

A Comparison of Two-Hybrid
Approaches for Detecting
Protein–Protein Interactions
J. Mehla1, J.H. Caufield, N. Sakhawalkar, P. Uetz1
Center for the Study of Biological Complexity, Virginia Commonwealth University, Richmond, VA,
United States
1
Corresponding authors: e-mail address: [email protected]; [email protected]

Contents
1. Introduction 334
2. The Bacterial Two-Hybrid System 335
2.1 Overview of the Bacterial Two-Hybrid System 335
2.2 Bacterial Two-Hybrid Protocol 336
3. The Yeast Two-Hybrid System 343
3.1 Overview of the Yeast Two-Hybrid System 343
3.2 Yeast Two-Hybrid Protocol 344
4. Comparison of Yeast and Bacterial
Two-Hybrid Methods 351
4.1 Comparison of Method Usage 351
4.2 Experimental Comparison of Bacterial and Yeast Two Hybrid Methods 353
4.3 Comparison of Method Strengths and Weaknesses 355
5. Conclusion 357
References 357

Abstract
Two-hybrid systems are one of the most popular, preferred, cost effective, and scalable
in vivo genetic approaches for screening protein–protein interactions. A number of
variants of yeast and bacterial two-hybrid systems exist, rendering them ideal for mod-
ern, flexible proteomics-driven studies. For mapping protein interactions at genome
scales (that is, constructing an interactome), the yeast two-hybrid system has been
extensively tested and is preferred over bacterial two-hybrid systems, given that users
have created more resources such as a variety of vectors and other modifications. Each
system has its own advantages and limitations and thus needs to be compared directly.
For instance, the bacterial two-hybrid method seems a better fit than the yeast two-
hybrid system to screen membrane-associated proteins. In this chapter, we provide
detailed protocols for yeast and bacterial two-hybrid systems as well as a comparison
of outcomes for each approach using our own and published data.

Methods in Enzymology, Volume 586 # 2017 Elsevier Inc. 333

ISSN 0076-6879 All rights reserved.
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334 J. Mehla et al.

1. INTRODUCTION
Most of the key biological processes of the cellular machinery, includ-
ing transcription, translation, protein quality control, transport, and signal-
ing are dependent on protein–protein interactions (PPIs) that occur either
individually or in multiprotein complexes. For example, the 26S proteasome
requires interactions among more than 30 different proteins organized in
three subcomplexes. Together, these proteins serve a common set of
functions.
The mapping of PPIs on a genome or proteome scale remains challeng-
ing and has been comprehensively explored only in a few model organisms
such as Escherichia coli, budding yeast, and humans. Numerous biochemical
and genetic approaches have been developed to study PPIs. This review
focuses on the yeast (Y2H) and bacterial (B2H) two-hybrid methods, which
are among the most explored in vivo genetic tools to map PPIs. The full
extent of biochemical and genetic approaches in use is beyond the scope
of this chapter but several detailed surveys have been published (e.g.,
Kuhn et al., 2014; Rao, Srinivas, Sujini, & Kumar, 2014; Syafrizayanti,
Betzen, Hoheisel, & Kastelic, 2014).
Both Y2H and B2H are methods that have been used to map and iden-
tify PPIs networks in many organisms (Mehla et al., 2015; Wang et al.,
2010). They can be used on a small-scale or as high-throughput screening
approaches. Two-hybrid methods are based on the principle of restoring
protein activity upon noncovalent reconstitution of split protein fragments.
One fragment of a modular protein is fused to a protein X, and the other
fragment is fused to a protein Y. The resulting fusion proteins may be
referred to as a bait and prey. If the proteins X and Y interact upon
coexpression, the modular protein is reconstituted, regains its activity,
and its activity is detected through a reporter gene. Although both the
B2H and Y2H work on similar principles, there are fundamental differ-
ences and both have distinct advantages and limitations. Different users
may choose one over another depending on their needs, available
resources, and properties of the proteins to be screened, and ease of screen-
ing. This chapter describes a detailed protocol for high-throughput yeast
and bacterial two-hybrid methods and a comparison of their use and some
typical results.
A Comparison of Two-Hybrid Approaches 335

2. THE BACTERIAL TWO-HYBRID SYSTEM

2.1 Overview of the Bacterial Two-Hybrid System
The B2H system may be used as an alternative to Y2H screening. The B2H
system has been applied to screening of proteins from different sources
(bacterial, mammalian, and viral), of different size and cellular location
(cytoplasmic, peripheral, and inner membrane), and of different functions
(transporters, enzymes, regulatory proteins, etc.). Although we do not know
of any systematic comparison of the two systems, the B2H may be preferable
to Y2H when screening bacterial proteins as the screening conditions are
more biologically similar to the proteins’ natural environment.
The most commonly used B2H system is the bacterial adenylate cyclase-
based two-hybrid system (BACTH) which is based on the reconstitution of
adenylate cyclase activity in E. coli (Fig 1). The catalytic domain of bacterial
adenylate cyclase has two subdomains: a 25 kDa fragment (T25 or cyaAT25)

Fig. 1 Principle of the bacterial two-hybrid system. The T18 and T25 domains of ade-
nylate cyclase are shown in different conditions. The intact adenylate cyclase domains
produce residual cAMP synthesis. The segregated T18 and T25 domains do not produce
cAMP, and hence there is no observed phenotypic change. The interacting proteins
X and Y can bring together T18 and T25 domains of adenylate cyclase in close proximity
and restore cAMP synthesis, leading to characteristic phenotypes on indicator plates.
336 J. Mehla et al.

with a catalytic site and a 18 kDa fragment (T18 or cyaAT18) with a binding
site for calmodulin. These domains are not forming a functional protein
when they are physically separated. The test proteins (X and Y) are fused
to the T25 and T18 domains, respectively, allowing interactions between
them to bring the domains in close proximity and—in the presence of
calmodulin—restore adenylate cyclase activity. The restored activity leads
to cyclic adenosine monophosphate (cAMP) synthesis. The cAMP pro-
duced by the reconstituted adenylate cyclase forms a complex with catabo-
lite activator protein, inducing the transcriptional activation of catabolic
operons such as lactose or maltose and yielding a characteristic phenotype
in a cya
E. coli strain (usually BTH101/DHM1) on selective or indicator
media plates.

2.2 Bacterial Two-Hybrid Protocol

2.2.1 Bacterial Strains and Vectors
Bacterial two-hybrid screens as described by Karimova, Pidoux, Ullmann,
and Ladant (1998) frequently employ E. coli BTH101 and/or DHM1 as
reporter strains. Both strains are adenylate cyclase deficient but only the latter
of these two strains contains a recA1 mutation, potentially improving plasmid
stability. Store these stocks at –80°C.

2.2.1.1 Genotypes
BTH101: F
, cya-99, araD139, galE15, galK16, rpsL1, hsdR2, mcrA1,
mcrB1
DHM1: F
, cya-854, recA1, endA1, gyrA96, thi1, hsdR17, spoT1, rfbD1,
glnV44(AS)
As described earlier, bacterial two-hybrid screens use vectors allowing for
expression of hybrid proteins containing either T25 or T18 adenylate cyclase
fragments. The vectors pKT25 (Kanr, cyaAT25 fragment) and pUT18
(Ampr, cyaAT18) are available in several variants (see Table 1).
Genes encoding the proteins of interest (X and Y) are amplified by PCR
and cloned by traditional or recombination cloning (e.g., using the Gateway
system, Hartley, 2000). The latter method is preferred for preparation of
clone sets as it can be performed with higher cloning efficiency than tradi-
tional cloning. With Gateway cloning, the attL-flanked ORFs can be rec-
ombined into the attR-flanked BACTH-DEST plasmids (pST25 and
pUT18C) using the LR reaction to generate attB-flanked ORFs within
the BACTH vectors. Expression clones may be prepared with the vectors
pST25, pSTM25, pUT18, and pUTM18, among others. Outer membrane
Table 1 Vectors Frequently Used in Two-Hybrid Screens
Type Fusion Selection
of Fusion
Assay Vector Promoter Protein DBD/T18 AD/T25 Yeast Bacterial Ori Source
Y2H Baits pGBKCg t-ADH1 Gal4 C-terminus – Trp1 Kanamycin 2μ Stellberger et al.
(2010)
pGBGT7g t-ADH1 Gal4 N-terminus – Trp1 Gentamycin 2μ Rajagopala et al.
(2009, 2010)
Preys pGADCg fl-ADH1 Gal4 – C-terminus Leu2 Ampicillin 2μ Stellberger et al.
(2010)
pGADT7g fl-ADH1 Gal4 – N-terminus Leu2 Ampicillin 2μ Chen et al. (2010)
B2H pKT25 pLac Adenylate – N-terminus – Kanamycin p15A Karimova et al.
cyclase (1998)
domain
pKNT25 pLac Adenylate – C-terminus – Kanamycin p15A Karimova et al.
cyclase (1998)
domain
pUT18C pLac Adenylate C-terminus – – Ampicillin ColE1 Karimova et al.
cyclase (1998)
domain
pUT18 pLac Adenylate N-terminus – – Ampicillin ColE1 Karimova et al.
cyclase (1998)
domain
Continued
Table 1 Vectors Frequently Used in Two-Hybrid Screens—cont’d
Type Fusion Selection
of Fusion
Assay Vector Promoter Protein DBD/T18 AD/T25 Yeast Bacterial Ori Source
pST25 pLac Adenylate – N-terminus – Spectinomycin p15A Ouellette et al.
cyclase (2014)
domain
pSNT25 pLac Adenylate – C-terminus – Spectinomycin p15A Ouellette et al.
cyclase (2014) and Battesti
domain and Bouveret
(2008, 2012)
pSTM25 pLac Adenylate – N-terminus – Spectinomycin p15A Ouellette et al.
cyclase (2014) and Battesti
domain and Bouveret
(2008, 2012)
pUTM18C pLac Adenylate C-terminus – – Ampicillin ColE1 Ouellette et al.
cyclase (2014) and Battesti
domain and Bouveret
(2008, 2012)
Gateway- pST25- pLac Adenylate – N-terminus – Spectinomycin p15A Ouellette et al.
compatible DEST cyclase (2014) and Battesti
vectors domain and Bouveret
(2008, 2012)
pSNT25- pLac Adenylate – C-terminus – Spectinomycin p15A Ouellette et al.
DEST cyclase (2014)
domain and Battesti and
Bouveret (2008,
2012)
pUT18C- pLac Adenylate C-terminus – – Ampicillin ColE1 Ouellette et al.
DEST cyclase (2014) and Battesti
domain and Bouveret
(2008, 2012)
pSTM25- pLac Adenylate – N-terminus – Spectinomycin p15A Ouellette et al.
DEST cyclase (2014) and Battesti
domain and Bouveret
(2008, 2012)
pUTM18C- pLac Adenylate C-terminus – – Ampicillin ColE1 Ouellette et al.
DEST cyclase (2014) and Battesti
domain and Bouveret
(2008, 2012)
340 J. Mehla et al.

(OM) bound, peripheral or extracytoplasmic proteins should be transferred

to pSTM25 or pUTM18 B2H expression vectors. These modified vectors
have an extra transmembrane segment fused downstream to T25 and T18
cyclase domains to detect interactions for OM or peripheral proteins
(Ouellette, Gauliard, Antosov, & Ladant, 2014). The Gateway vectors
should be propagated in ccdB resistant E. coli strains at 30°C.

2.2.2 Reagents and Media

E. coli cultures may be grown with LB media. For LB/X-Gal indicator
plates, add X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside)
to 40 μg/mL. IPTG may also be included in these plates (at 0.5 mM) to
improve protein expression and hence observation of interactions.
MacConkey agar with 1% maltose may also be used for indicator plates.
Where specified, ampicillin (Amp) and spectinomycin (Spc) should each
be used at 100 μg/mL.
One liter of Z-buffer is prepared as follows:
16.1 g Na2HPO4 7H2O (or 8.5 g anhydrous)
5.5 g NaH2PO4 H2O
0.75 g KCl
0.246 g MgSO47H2O
Add H2O to the final volume and adjust to a pH of 7 if needed. Store
at 4°C.
Add 2.7 μL β-mercaptoethanol (β-ME) per mL of Z-buffer immediately
before use in an assay. Retain a portion of Z-buffer without β-ME added
(see review by Battesti & Bouveret, 2012 for further details.)
ONPG (2-nitrophenyl β-D-galactopyranoside) is used to assay β-galactosidase
activity and hence the strength of a protein interaction. Solutions may be pre-
pared as 4 mg/mL in Z-buffer and should be prepared fresh on the day of each
assay.
This procedure also requires the following solutions: 0.1% SDS, chloro-
form for cell lysis, and 1 M Na2CO3 to stop the enzymatic reaction.

2.2.3 Bacterial Two-Hybrid Procedure

See Fig. 2 for a summary of this procedure.

2.2.3.1 Cotransformation of Expression Constructs Into Reporter Strains

Expression constructs of test proteins encoding the T25-X (or X-T25) and
T18-Y (or Y-T18) fusions are cotransformed into competent BTH101
E. coli. Competent cells can be prepared on the same day as transformation.
A Comparison of Two-Hybrid Approaches 341

Fig. 2 Stepwise overview of the bacterial two-hybrid procedure. The test fusion pro-
teins X (bait) and Y (prey) are constructed and cotransformed in the E. coli BTH101 strain.
The interaction between X and Y can restore the cAMP synthesis, leading to the
observed phenotypes on indicator plates: for example, red and blue colonies on
MacConkey/maltose and LB/X-Gal plates, respectively.

Routine heat shock-based transformation protocols are ideal, especially for

high-throughput screens. See Chung, Niemela, and Miller (1989) for a basic
protocol.
1. Transfer 30 μL of competent cells per reaction to prechilled microce-
ntrifuge tubes or 96-well plate(s) on ice.
2. Add 100 ng of both expression constructs (both bait and prey) to the
cells.
3. Keep cells on ice for at least 30 min. Heat shock at 42°C for 45 s. After
the heat shock, return the cells to ice for 5 min.
342 J. Mehla et al.

4. Add 100 μL of sterile LB broth to each tube or well and incubate at 37°C
for 45–60 min.
5. Plate the cells on selective agar plates (e.g., LB/Amp/Spc) and incubate
at 30°C for 24–36 h.

2.2.3.2 Screening Cotransformants for Interactions

Select the cotransformants for further screening on indicator plates at
30°C.
Indicator or selective media plates can be used to detect positive inter-
actions between test proteins. The positive interactions should produce
specific phenotypes on respective indicator plates, i.e., blue colonies on
LB-X-Gal or red on MacConkey-maltose. This may take up to 4 days. If
no interaction (negative result) occurs, all colonies should be colorless on
indicator plates. This method can be scaled up for screening libraries.
The same batch of cells can be used for both plating on indicator plates
and for quantification using the β-galactosidase assay.

2.2.3.3 Quantification of PPIs With the β-Galactosidase Assay

The interactions between proteins (X and Y) can be quantified by measuring
β-galactosidase activity in liquid cultures using the classical Miller assay
(Miller, 1972, 1992). The β-galactosidase activity is usually expressed in
Miller units (one unit corresponds to 1 nmol of ONPG hydrolyzed per
min at 28°C) per mg of bacterial dry weight.
This protocol is used to determine the level of β-galactosidase activity in
a bacterial two-hybrid interaction screen and may be scaled up for high-
throughput use in 96-well plate format. The level of galactosidase activity
will vary depending on the level of interaction for strongly and weakly inter-
acting proteins. Thus, we can quantify the strength of an interaction
between two proteins. The time required for the assay to develop reliable
readings/color can vary depending on the strength of the interactions, espe-
cially if the interaction is very weak.
Prepare overnight cultures of each cotransformed culture to be assayed.
Transfer cultures to a 96-well plate after their overnight growth. Set an incu-
bator or water bath to 30°C.
1. Use 0.1 mL of each overnight culture to determine the absorption at
OD600 with a plate reader.
2. Collect the cells in the plate by centrifugation at 2000–4000 rpm in a
benchtop centrifuge (at least 1000  g) for 5–10 min.
A Comparison of Two-Hybrid Approaches 343

3. Remove the supernatant and resuspend each cell pellet in 80 μL of

Z-buffer (with β-ME).
4. Add 10 μL of chloroform to each well.
5. Add 10 μL of 0.1% SDS to each well.
6. Vortex each plate, gently, for 30 s.
7. Centrifuge the lysate as in step 2.
8. Transfer 0.1 mL of each lysate to a clean 96-well plate. Ensure
each lysate is clear and that no turbidity is transferred to the new
plate.
9. Prewarm the ONPG solution to 30°C. The plate can also be pre-
incubated at 30°C for 5 min to equilibrate the temperature of the
suspension.
10. Start the reaction by adding 20 μL of ONPG (4 mg/mL) to each well.
Record the start time.
11. Monitor the reaction at 30°C until you see a yellow color (similar to
that of LB media) develop or the reaction has run for 90–120 min.
12. Stop the reaction by adding 30 μL of a 1 M Na2CO3 solution. Record
the reaction stop time.
13. Determine the OD420 Abs of each well using a plate reader.
14. Convert the OD420 into Miller units (see Miller, 1992) using the fol-
lowing conversion:

Miller Units ¼ 1000  ðOD420 =ðOD600 of culture  culturevolume

reactiontimeð min ÞÞÞ

3. THE YEAST TWO-HYBRID SYSTEM

3.1 Overview of the Yeast Two-Hybrid System
The Y2H assay is an in vivo genetic method developed by Fields and Song
(1989) to screen for binary PPIs.
In this method, the yeast transcription factor Gal4 is split into a DNA-
binding domain (DBD) and an activation domain (AD). Protein pairs are
fused to the DBD and AD, and the resulting DBD-X and AD-Y fusions are
generally referred to as bait and prey. Only the simultaneous coexpression
of both the bait and an interacting prey will reconstitute the full transcrip-
tion factor and permit transcription (Fig. 3).
344 J. Mehla et al.

Fig. 3 Principle of the yeast two-hybrid system. The segregation of Gal4 DNA-binding
domain (DBD) and activation domain (AD) prevents transcription of the reporter gene,
leading to a lack of colony growth. The interaction between bait and prey proteins
brings together the Gal4 DBD and AD and reconstitutes the transcription factor. This
leads to transcription of a reporter gene (i.e., HIS3) and results in yeast colony growth.

The subjects of a two-hybrid screen may be random libraries, such as frag-

ments of genomic or cDNA, or defined clone sets, usually in array format.
Arrays are preferred as they are easily scalable (i.e., a full 96-well plate of expres-
sion constructs can be screened against one protein of interest on a single plate)
and do not require sequencing to verify the identity of each interactor. Using
arrays may also help to decrease the incidence of false positive results.

3.2 Yeast Two-Hybrid Protocol

3.2.1 Yeast Strains and Vectors
The following protocol uses the yeast strains AH109 (for baits) and Y187 (for
preys) for high-throughput yeast two-hybrid screens. These strains are fre-
quently used and grow consistently, especially when provided with supple-
mental adenine in media (up to 100 mg/L).

3.2.1.1 Genotypes
AH109: MATa, trp1-901, leu2-3, 112, ura3-52, his3-200, gal4Δ, gal80Δ,
LYS2::GAL1UAS-GAL1TATA-HIS3, GAL2UAS-GAL2TATA-ADE2,
URA3::MEL1UAS-MEL1TATA-lacZ (James, Halladay, & Craig, 1996).
Y187: MATα, ura3-52, his3-200, ade2-101, trp1-901, leu2-3, 112, gal4Δ,
met
, gal80Δ, URA3::GAL1UAS-GAL1TATA-lacZ (Harper, Adami,
Wei, Keyomarsi, & Elledge, 1993).
A Comparison of Two-Hybrid Approaches 345

3.2.2 Reagents and Media

The selection mechanism used for diploid cultures in yeast two-hybrid
screens will depend upon the strains and vectors in use. Many methods
employ nutritional selection, i.e., if the two yeast strains are auxotrophs
for leucine and tryptophan, the yeast two-hybrid vectors must contain
complementing genes, e.g., LEU2 and TRP1, and diploid cultures must
be isolated on media lacking both amino acids. For yeast two-hybrid screens
using HIS3 as a reporter gene, selective yeast media may be supplemented
with 3-AT (3-amino-1,2,4-triazole) at concentrations between 1 and
50 mM. 3-AT acts as a competitive inhibitor of the HIS3 enzyme
(imidazoleglycerol-phosphate dehydratase) which will, therefore, reduce
background growth, allowing for easier distinction of positive colonies
from background.
Selective solid yeast medium, in single-well plates (e.g., Nunc
Omnitrays) (including -L, -T, -LT, and -LTH media)
10 TE buffer (0.1 M Tris–HCl, 10 mM EDTA), pH 7.5
YEPDA medium is prepared as:

Yeast extract 1%
Peptone 2%
Dextrose 2%
Adenine hemisulfate salt 0.01%

Sterilize by autoclaving. For solid medium, include 1.6% agar and pour
40–50 mL of sterile medium into each single-well Omnitray plate.
Allow to solidify.

3.2.3 Yeast Two-Hybrid Procedure

See Fig. 4 for a summary of this procedure.

3.2.3.1 Materials
These following methods have been designed to be performed using 96-well
plates (or 96-colony plates). The protocol may be used with manual colony
transfer or in high-throughput with the aid of a laboratory automation system,
such as a Beckman Coulter Biomek 2000, FX or FXP, along with a high-
density plate replicator attachment (e.g., 96/384 HDR Tool Body, V&P
Scientific, San Diego, CA) and compatible pins (“thin” pins of
0.381–0.457 mm or “thick” pins of 1.143–1.58 mm in diameter). Plates
346 J. Mehla et al.

Fig. 4 Stepwise overview of the yeast two-hybrid procedure. The test protein constructs
(bait and prey) are transformed into haploid yeast strains of opposite mating type (a and α).
The mating of two haploid strains carrying bait and prey plasmids produce the diploid
cells. Diploid cells are selected by transferring the mated array to double-dropout
(e.g., -LT) medium followed by selection of positive interaction by transferring cells
to triple-dropout (e.g., -LTH) medium.

may also be replicated manually using a 96-pin or 384-pin plate replicator.

Solid agar media should be prepared in single-well 86  128 mm Omnitray
microtiter plates (Thermo Fisher/Nunc).
Plate replicator pins must be sterilized between each pinning or they may
introduce cross-contamination. Rinse the tips of each pin in 20% bleach
(60 s), sterile dH2O (20 s), ethanol (95%, v/v; 60 s), and sterile dH2O (20 s).

3.2.3.2 Screening Protein Interactions Using Genomic or cDNA Libraries

Two-hybrid methods traditionally define screened fusion proteins as baits
and preys. This distinction is ensuring that the two vectors contain different
A Comparison of Two-Hybrid Approaches 347

genes for selection. Otherwise, nearly any protein may be used as a bait
or prey.
Some ORF collections and libraries provide sequences (including, in
some cases, nearly complete genomes) in two-hybrid vectors. These vectors
may require subcloning or a recombination-based cloning procedure (e.g.,
Gateway cloning) to produce a full set of expression vectors for two-hybrid
screens. ORFeomes are available for viral genomes (e.g., herpesviruses; Titz
et al., 2006), several bacterial genomes (e.g., E. coli; Rajagopala et al., 2010)
and some eukaryote species (e.g., Caenorhabditis elegans (Lamesch et al.,
2004), human (Rual et al., 2004), and Arabidopsis thaliana (Gong et al., 2004).
If a bait protein is screened against a random genomic (or cDNA) library
of prey proteins in a library screen identification of the interacting prey pro-
teins requires isolation and sequencing of positive clones. For further tech-
nical details, see Mehla et al. (2015).

3.2.3.3 Preparations and Array Construction

Arrays have certain advantages for Y2H screens, especially to validate the
positive interactions in a library screen (Mehla, Caufield, & Uetz, 2015a,
2015b). Because certain baits may self-activate (i.e., produce positives with-
out an interactions), usually the preys are arrayed in a defined order before
the actual Y2H screen or retest. Prey proteins usually do not generally pro-
duce self-activation without a bait protein.
For genome-wide screens, automation is strongly recommended, given
that manual work is not only straining, and error prone, but also time con-
suming and tedious. However, automation can be an expensive option.
Collaborating with a lab already in possession of automation equipment
may be an alternative.
The ORFs from entry vectors are assembled into Gateway-compatible
prey or bait vectors by Gateway cloning. The Gateway-compatible bait and
prey vectors are shown in Table 1. The prey and bait vectors are transformed
into mating-competent, haploid yeast strains Y187 (mating type “α”), and
AH109 (mating type “a”), respectively.

3.2.3.4 Yeast Transformation

This method is optimized for high-throughput transformation of bait or
prey plasmid clones into respective yeast strains and can be scaled up and
down as required.
1. Inoculate 10–20 mL of YEPDA with the haploid yeast strain and grow
overnight in a shaking incubator (200 rpm) at 30°C.
348 J. Mehla et al.

2. Dilute the cells in 100 mL (or as required) of fresh YEPDA to an OD600

of about 0.2–0.3. Grow cells for approximately 3–4 h at 30°C with
shaking. OD600 should be between 0.5 and 0.8 (or, at least one dou-
bling) such that the cells are in log phase of growth.
3. Centrifuge the culture in 50-mL sterile tubes and wash them with
25–50 mL sterile dH2O.
4. Then resuspend the cells in 1 mL of freshly-made 0.1 M LiAc/TE and
incubate on ice for 15 min followed resuspending again in 1 mL of
0.1 M LiAc/TE in 1.5-mL microcentrifuge tube on ice for another
15–20 min.
Prepare fresh 0.1 M LiAc/TE (1 mL of 1.0 M LiAc + 1 mL 10 TE +
8 mL of dH2O).
5. Prepare a transformation mix in a sterile 50-mL centrifuge tube (for
100 transformation reactions) as: 10 mL of 40% PEG (10 mL of 44%
PEG + 1 mL of 0.1 M LiAc/TE), 250 μL of 10 mg/mL carrier DNA
in a 50-mL tube and vortex vigorously followed by 1–2-mL of freshly
prepared competent yeast cells (previous step). Vortex again for 60 s.
6. Add 100 μL of transformation mix into a 96-well plate using a multi-
channel pipette or robotic liquid handler. The volume in each well
should not exceed 120 μL per well. For fewer samples, 1.5-mL centri-
fuge tubes may be used.
7. Then add 100 ng of plasmid DNA to each well and mix well with
pipette tip. Include one negative (carrier DNA but no plasmid
DNA) and positive controls (containing empty vector).
8. Seal the 96-well plate with adhesive aluminum foil and Parafilm to
secure edges and protect against contamination.
9. Vortex the plate gently for 3–4 min and incubate at 30°C for 45 min
with gentle shaking (60 rpm).
Make sure there is no cross-well contamination.
10. Then, incubate the plate at 42°C in a water bath for 30 min. Ensure that
water is not entering the wells.
For transformation in microcentrifuge tubes, the incubation time at
42°C incubation may be reduced to 15 min.
11. Then, centrifuge the 96-well plate for 8–10 min (700  g, RT). Dis-
card the supernatant either by pipette or by inverting the plate carefully.
12. Add 50–100 μL sterile dH2O to each well and spread it on selective
media plates.
Make sure the plates are dry.
13. Incubate plates at 30°C for at least 2–3 days.
A Comparison of Two-Hybrid Approaches 349

Strains, such as Y187, may require 3–4 days to yield noticeable transformant
growth.
The specific transformants (each carrying one specific prey vector), are
arrayed on selective media plates in a 96- or 384-format in duplicates or qua-
druplicates. For freeze stocks, the arrays may be cultured in rich (YEPDA) or
selective liquid media, resuspended in 20% glycerol, and stored at –80°C for
long-term storage. For routine use, the arrays can be stored on solid selective
medium for up to 3 months at 4°C and fresh arrays can be propagated using
storage plates prior to Y2H screen.
For long-term storage, the master prey array should be kept on selective
plates (e.g., -Leu plates). The master copy of the array should only be used to
make fresh copies on YEPDA agar plates for mating. Copying the array onto
fresh selective plates every 2 weeks should protect against plasmid loss and
contamination.

3.2.3.5 Bait Auto Activation Test

The bait auto activation test is recommended prior to Y2H screen and is
performed to measure the background potential of baits to activate transcrip-
tion of the reporter gene (here: HIS3) in the absence of prey protein (or pres-
ence of empty prey vector). This will help to optimize actual Y2H screen
conditions, saving time and resources.
(1) Plate bait clones on YEPDA media or selective media in a single-well
Omnitray or Petri plate in a standard 96 or 384-spot format depending
on the number of clones.
(2) The bait strains are mated with a prey strain carrying the prey empty
vector (i.e., Y187 strain with pGADCg empty vector) on a YEPDA
plate at 30°C.
(3) Select diploid cells on appropriate selective media (i.e., -LT, see above)
at 30°C.
(4) Transfer the diploid colonies to selective media (i.e., -LTH, see earlier)
with and without 3-AT for selecting the HIS3 reporter gene activity.
3-AT concentrations of 0, 1, 3, 5, 10, and 25 mM are recommended for
an initial screen. Almost all background should be gone within this
range. However, in special circumstances concentrations up to 100 mM
may be required.
(5) Incubate the selective plates at 30°C for up to 1 week. Record the low-
est 3-AT concentration suppressing background growth for each bait
that should be added to selective plates in the actual Y2H screens.
350 J. Mehla et al.

3.2.3.6 Screening for Protein Interactions

After the bait self-activation test, the actual Y2H screens are performed as
described later.
1. Use the sterile pin replicator to transfer the yeast prey array from selective
plates to single-well microtiter plates containing solid YEPDA medium
and grow the array overnight in a 30°C incubator. Alternatively, the
fresh prey array can be made in YEPDA broth in a 96-well plate.
2. Inoculate 15–20 mL of YEPDA broth medium in a 50-mL sterile
tube with a bait strain, overnight in a shaking incubator (at 200 rpm)
at 30°C.
If the bait strains are frozen, they should be activated prior to exper-
iment by pinning them on rich media and followed by pinning on selec-
tive solid medium plates. Overnight grown baits and preys culture may
increase mating efficiency.
3. Pin the overnight bait cells onto solid YEPDA media in an Omnitray
plate by dipping the sterile pins in bait liquid culture. Replicate as
required and allow the spots to dry for 15–20 min.
YEPDA plates should be free of any moisture before pinning cells.
4. Transfer overnight prey array cells onto bait spots. Touch the prey cells
with sterilized pins and transfer them onto the plated bait spots. Incubate
for 36–48 h at 30°C to allow mating.
Addition of adenine in the culture media may increase the mating
efficiency of some baits.
5. Select diploid cells by transferring the colonies from mating plates to
double-dropout selective medium plates (i.e., -LT) using the sterilized
pinning tool. Grow the cells at 30°C for 2–3 days or until the colonies
are 1 mm in diameter.
Only the diploid cells containing both the prey and bait vectors will
grow on -LT plates. Low mating efficiency will reduce the quality and
coverage of Y2H screen results.
6. Transfer the colonies from -LT plates to screening media plates (i.e.,
-LTH, with or without 3-AT) using the sterilized pinning tool. The
required concentration of 3-AT will depend on the bait autoactiva-
tion test (see steps 16 through 20). Incubate the plates at 30°C for
4–7 days.
7. Score the positive interactions by counting colonies that are significantly
large in size compared to background.
The screening plates should be monitored daily for positive colonies.
Typical positive interactions should appear within 3–5 days.
A Comparison of Two-Hybrid Approaches 351

3.2.3.7 Retesting of Positive Interactions

The main concern associated with the Y2H screen is the possibility of a large
number of false positives. The use of different vectors and retesting of all the
positive interactions through repeated mating can help to reduce the num-
ber of false positives. Only the reproducible interactions in retest Y2H
screens will be counted as positive interactions. Moreover, screening the
same baits and preys using multiple vectors provides additional evidence
for positive interactions. Alternative methods are also available for confirma-
tion of Y2H interactions (for protocols, see Rajagopala, Titz, & Uetz, 2007;
Serebriiskii, Golemis, & Uetz, 2005).

4. COMPARISON OF YEAST AND BACTERIAL

TWO-HYBRID METHODS
4.1 Comparison of Method Usage
The Y2H screens can capture only direct pairwise interactions, including
weak interactions. Y2H screens are a heterologous system for bacterial pro-
teins which comes with some limitations. For example, nonphysiological
level of protein expression, absence of necessary cofactors, ligands or chap-
erones for functional activity, and issues with the translocation of bacterial
proteins to the yeast nucleus. This leads to low sensitivity and coverage
and up to 70% of PPIs are not detected even in comprehensive screens.
The specificity of the Y2H screens is enhanced with the advancement of
multivector and next-generation Y2H screens for mapping membrane
interactomes (Lam & Stagljar, 2012; Stellberger et al., 2010).
The B2H methods are developed to be performed in their natural host,
and thus can detect both direct and indirect protein interactions. The
adenylyl cyclase-based B2H is the most commonly used B2H technique
and is suitable for detecting interactions between cytoplasmic, membrane
proteins, and periplasmic proteins. Most of the B2H studies are performed
at small scale. Traditionally, the Y2H system has been the preferred choice
over B2H, for detecting PPIs on genome-wide scale. However, there is no
objective reason why the B2H should not be applicable on a large-scale.
Two-hybrid methods are popular fixtures in the biomedical literature, as
a chronological search of PubMed reveals (Fig. 5). Here, we show the total
number of publications per year containing the phrase “two-hybrid” any-
where in their title or abstract. At its peak in the early 2000s, two-hybrid
methods were highlighted in more than 900 publications each year, with
yeast two-hybrid approaches providing the majority of those methods.
352 J. Mehla et al.

Fig. 5 Count of publications in PubMed featuring two-hybrid methods. Counts include

all publications including the phrases shown in the key at right in their title or abstract.
Counts made in July 2016; year to date values for 2016 are shown at far right.

Fig. 6 Count of citations to Fields and Song (1989). Counts include all citations to the
Fields and Song paper in Nature describing their yeast two-hybrid system as of July
2016. Citation counts are from Web of Science; the publication has been cited
4169  in total by this metric.

Bacterial two-hybrid methods have been comparatively less popular but

have been modified to be used in concert with other methods
(Serebriiskii et al., 2009) or to more easily confirm expression of the desired
fusion proteins (Battesti & Bouveret, 2008). Some of the difference in pop-
ularity between yeast and bacterial two-hybrid methods may simply be due
to the time since their introduction: Karimova et al. did not publish details of
their bacterial two-hybrid method until 1998, nearly a decade after Fields
and Song described Y2H (see Fig. 6 for counts of citations per year to this
paper alone). It should also be noted that two-hybrid methods of any type
may not always serve as a project’s primary data source: 370 publications
A Comparison of Two-Hybrid Approaches 353

Fig. 7 Count of interactions in IntAct by identification method. Counts include all inter-
actions in the IntAct database of protein interactions as of August 2016. “Two-hybrid
interactions” include those identified with any two-hybrid, two-hybrid array, or two-
hybrid pooling method and all variations. “Pull-down interactions” are those identified
with any pull-down method, not including method variations. Each square represents
10,000 interactions.

from 2015 in PubMed included bacterial two-hybrid methods though just

40 in the same year mentioned the method in their title or abstract. Some
publications may use two-hybrid approaches to generate further experimental
evidence and may, therefore, avoid discussing the method in detail. Though
perhaps increasingly seen as a basis for new approaches rather than a core meth-
odology, two-hybrid methods remain prominent among PPI screens.
Likely owing to their popularity and ease of implementation, two-hybrid
methods have not only remained popular approaches but also have contrib-
uted more than a third of all interactions in the IntAct molecular interaction
database (Fig. 7). These counts include all interactions in the database—even
if the interaction was studied by more than one group or with more than one
method—and therefore serve as a summary of publicly available interaction
data and the methods responsible. For some species, two-hybrid methods
are especially popular: more than half of the PPIs in IntAct among bacterial
proteins were identified using two-hybrid approaches.

4.2 Experimental Comparison of Bacterial and Yeast Two

Hybrid Methods
The main concerns of two-hybrid methods are the rate of false positives and
the bait self-activation especially for Y2H screens. Despite its extensive use,
two-hybrid methods have rarely been explored simultaneously or in parallel.
To better understand the experimental outcomes of B2H and Y2H
354 J. Mehla et al.

methods, an extensive study should be done to compare different types of

vector, types of proteins, and parameters affected by the host. The types
of proteins may be further classified in terms of their functions and location.
More extensive studies are required to comprehend the outcomes and suit-
ability of two-hybrid methods. We tried to get an idea of the outcomes of
B2H and Y2H screens by screening the same set of interacting proteins by
both B2H and Y2H (Fig. 8). The protein “X” was tested against a set of
12 prey partners by both methods. Few positive interactions are captured

Fig. 8 Experimental examples and comparison of results from yeast and bacterial two-
hybrid screens. A representative high-throughput B2H screen is shown here (only a part
of 96-well plate). The blue and red color colonies on LB/X-Gal and McConkey/maltose
indicator plates are considered as positive interactions (A). The same set of test protein
pairs were tested for interactions by both B2H and Y2H methods (B and C). The red color
and visible growth of colonies are considered as positives in B2H and Y2H screens,
respectively. Few positive interactions are detected in the B2H screens. However, no
single interaction was captured in the Y2H screens as discussed in the text earlier.
A Comparison of Two-Hybrid Approaches 355

in our B2H screens, however, not even a single interaction was detected by
the Y2H method. There can be several reasons for this result. The most
plausible explanation is that the fusion proteins in both systems are suffi-
ciently different to allow an interaction in one system but not the other.
Rajagopala, Hughes, and Uetz (2009) as well as Chen, Rajagopala,
Stellberger, and Uetz (2010) have shown that small differences even within
the Y2H can cause substantial outcomes in the set of detected PPIs. Given
that only a fraction of all interactions are detected in each screen, this
explains even nonoverlapping resulting PPIs in different screens. Most of
the proteins selected in our example are membrane proteins which is
another confounding reason that not a single interaction was captured in
the Y2H screen. Also, the function of the interacting proteins might have
some implications. For instance, directly or indirectly, the selected proteins
are predicted to be involved in the LPS assembly or outer membrane asym-
metry in bacteria, and thus could lead to lower mating efficiency and even
may cause toxicity in the yeast cells. The interactions detected by Y2H
screens took place in the nucleus, and thus, the membrane or outer mem-
brane proteins which are having hydrophobic transmembrane region/
domains could not be able to reach the nucleus and ultimately no interaction
(Auerbach & Stagljar, 2005). In B2H screens, the interactions are not
restricted to the nucleus and only the protein complementation is required
to detect the positive interactions. That could give the B2H screens a certain
advantage for screening trans-membrane (or even peripheral membrane)
proteins unless a specialized membrane yeast two-hybrid assay is used. That
is why the interactions shown here are captured only by B2H screens.
Also, the proper folding of the membrane proteins might need the phos-
pholipids bilayers, and thus can lead to false negatives or to no interaction in
Y2H screens. These limitations could be overcome by screening only the
cytoplasmic domain of the membrane proteins or using a low copy number
plasmid to avoid any toxicity (if any). Not only the localization of the pro-
teins causes false negatives but also the specific condition (e.g., oxidative,
cofactors, etc.) or posttranslational modification required for the proper
folding/activity of the proteins can lead to higher rate of false negatives.
Another reason for higher false negatives in Y2H screens could be the tox-
icity of interacting proteins upon overexpression (Br€ uckner, Polge, Lentze,
Auerbach, & Schlattner, 2009).

4.3 Comparison of Method Strengths and Weaknesses

Affinity purification coupled with mass spectrometry (AP–MS) is the most
popular biochemical method for detecting PPIs. In AP/MS, the tagged bait
356 J. Mehla et al.

proteins are used to pull out interacting prey proteins. The two-hybrid
methods are widely used and have produced large number of interactions
available in different databases (Fig. 7). Two-hybrid methods are preferred
because of the following:
(1) Accessibility: Two-hybrid methods can be carried out in a lab with
basic and inexpensive microbiological reagents and tools. They do
not require any specific equipment unless being carried out on a large
scale when expensive robots may be required.
(2) Type of interactions: Two-hybrid methods detect the in vivo binary
interactions between individual proteins. The interacting proteins
may not be from the same organism. They can be from two different
bacteria or a bacterium and its phage, etc. Entire proteins, protein
domains, or protein fragments can be used for screening interactions.
There is no size limit for proteins being screened.
(3) Sensitivity, scalability, and coverage: The assay is highly sensitive. Even
transient and weak interactions can be detected using two-hybrid
methods. Two-hybrid methods are easy to scale up and down.
However, several features of two-hybrid assay impose limitations on the
type and nature of interactions that can be analyzed.
(1) False positives and self-activation of baits: Potentially high rate of false
positives is one of the major drawbacks of two-hybrid assays. However,
false positives can be reduced by including stringent experimental con-
ditions (e.g., higher 3-AT concentrations, using different vector com-
binations) and by filtering the resulting dataset (e.g., biological
relevance or plausibility of interactions). Another related problem is
self-activating baits. Self-activating baits activate the reporter gene
without interacting with the prey protein. The bait self-activation con-
sequences are not typically associated with B2H screening.
(2) Lack of detection of interactions for certain proteins. Proteins that can-
not fold intracellularly (e.g., membrane proteins) or cannot enter the
nucleus cannot be studied using transcription-based assays. Protein
fusions that are either toxic to or unstable in yeast (Y2H) or E. coli
(B2H) cannot be screened using two-hybrid methods. In general, all
interactions dependent on posttranslational events that do not occur
in yeast (Y2H) or E. coli (B2H) will not be detected. Two-hybrid
systems have transcription factor domains fused either to the N- or
C-terminus of proteins which may block interactions dependent on
a free N- or C-terminus.
A Comparison of Two-Hybrid Approaches 357

5. CONCLUSION
No single two-hybrid method is best suitable for every individual or
lab. The selection of methods for mapping PPIs is dependent on the needs,
and resources availability. A comprehensive analysis of these methods in
terms of nature of proteins, host, and methodology might help in planning
and choosing a better approach for mapping PPIs.

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