Two Hybrid Technologies: Methods and Protocols

METHODS I N MOLECULAR BIOLOGY™

Series Editor

John M. Walker

School of Life Sciences

University of Hertfordshire

Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:

http://www.springer.com/series/7651

Two Hybrid Technologies

Methods and Protocols

Edited by

Bernhard Suter

Max-Delbrück-Centrum für Molekulare Medizin, Quintara Biosciences, Albany, CA, USA

Erich E. Wanker

Max-Delbrück-Centrum für Molekulare Medizin, Berlin-Buch, Germany

ISSN 1064-3745 e-ISSN 1940-6029

ISBN 978-1-61779-454-4 e-ISBN 978-1-61779-455-1

DOI 10.1007/978-1-61779-455-1

Springer New York Dordrecht Heidelberg London

Library of Congress Control Number: 2011940814

© Springer Science+Business Media, LLC 2012

All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the

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Editors

Bernhard Suter

Max-Delbrück-Centrum

für Molekulare Medizin

Quintara Biosciences

Albany, CA, USA

[email protected]

Erich E. Wanker

Max-Delbrück-Centrum

für Molekulare Medizin

Berlin-Buch, Germany

[email protected]

v

Preface

Protein–protein interactions (PPIs) are strongly predictive of functional relationships

among proteins in virtually all processes that take place in the living cell. Therefore, the

comprehensive exploration of interactome networks is one of the major goals in systems

biology. The development of “interactomics” as a fi eld is largely driven by the development

of innovative technologies and strategies for effi cient screening, scoring, and validation of

PPIs. The aim of this book is to provide a compendium of state-of-the art-protocols for the

investigation of binary PPIs with the classical yeast two-hybrid (Y2H) approach, Y2H vari￾ants, and other in vivo methods for PPI mapping. Given the broad range of methodologies

currently available, biochemical approaches like proteome-wide co-immunoprecipitation,

and other in vitro and in vivo methodologies are not to be considered here. It needs to be

emphasized, however, that alternative methods are very important for the complementa￾tion and validation of Y2H screens.

The book is structured into two sections. The fi rst gives a survey of protocols that are

currently employed for Y2H high-throughput screens by different expert labs in the fi eld.

Rather than detailing the principles of screening, which have been described previously,

the focus is on different implementations of Y2H interactome mapping. First, two articles

by Peter Uetz review the most important developments and applications of Y2H high￾throughput screening. Then, Russ Finley, Ulrich Stelzl, Manfred Koegl, and coauthors

describe their automated screening procedures in detail. A view on interactome research

in pathogenic organisms is provided by Vincent Lotteau and Lionel Tafforeau (viral inter￾actomes), and Douglas LaCount (interactomes of malaria parasites). Xiaofeng Xin and

Thierry Mieg complement experimental protocols with their recently developed strategy

of smart-pooling by shifted transversal design. Two more articles deal with bioinformatics

for the analysis of Y2H data sets. Russ Finley and team discuss confi dence scoring, whereas

Gautam Chaurasia and Matthias Futschik describe the design of a database for high￾throughput Y2H data (UniHI, Max Delbrueck Centrum, Berlin). John Reece-Hoyes and

Albertha Walhout present a high-throughput yeast one-hybrid variant for the identifi ca￾tion of proteins that bind-specifi c DNA segments. Finally, contributors from the lab of

Young Chul Lee introduce their “one- plus two-hybrid system” for the effi cient identifi ca￾tion of PPIs altered by missense mutations.

The second part of the book considers innovative PPI detection methods that have the

potential to emerge as alternative high-throughput methodologies. An important future role

can be expected for systems that rely on the functional reconstitution (complementation) of

reporter proteins by fused bait and prey proteins. A chapter on the split-ubiquitin-based

system to screen for membrane protein interactions is provided by Igor Stagljar, whereas

Mandana Rezwan and Daniel Auerbach of Dualsystems Biotech AG describe an approach to

screen for interactors using the reconstitution of a split-TRP1 protein. For future human

interactome studies, procedures that can reconstitute PPIs directly in mammalian cells could

provide a better physiological context compared to yeast. A mammalian two-hybrid system

based on the tetracycline-repressor system is presented by Kathryn Moncivais and Zhiwen

vi Preface

Zhang. A different principle in mammalian cells is used by Heinrich Leonhardt and team in

their fl uorescent two-hybrid approach, where bait and prey proteins are recruited to specifi c

chromosomal locations. Perhaps the most advanced strategy for binary PPI mapping in

mammalian cell culture is the mammalian protein–protein interaction trap (MAPPIT),

developed by Jan Tavernier and his group. It is based on complementation of a cytokine

receptor complex operating in mammalian cells. In the high-throughput ArrayMAPPIT

application, prey proteins are arrayed in high-density microtiter plates to screen for interac￾tion partners using reverse transfection into a bait-expressing cell pool. A variation of

MAPPIT can be used to test substances that disrupt PPIs. Finally, Moritz Rossner provides

a protocol for the use of uniquely expressed oligonucleotide tags (EXTs) that integrate

complementation assays based on TEV protease and transcription factor activity profi ling.

Together, the protocols supply researchers with a comprehensive toolbox for the identifi ca￾tion of biologically relevant protein interactions.

We are very grateful to all contributing authors for their great commitment to this

project. We would like to express special gratitude to Dr. John M. Walker for his guidance

and continuous support during the preparation of the manuscript.

Albany, CA, USA Bernhard Suter

Berlin, Germany Erich E. Wanker

vii

Contents

Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

1 Matrix-Based Yeast Two-Hybrid Screen Strategies and Comparison of Systems . . . . 1

Roman Häuser, Thorsten Stellberger, Seesandra V. Rajagopala,

and Peter Uetz

2 Array-Based Yeast Two-Hybrid Screens: A Practical Guide . . . . . . . . . . . . . . . . . . . 21

Roman Häuser, Thorsten Stellberger, Seesandra V. Rajagopala,

and Peter Uetz

3 High-Throughput Yeast Two-Hybrid Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

George G. Roberts III, Jodi R. Parrish, Bernardo A. Mangiola,

and Russell L. Finley Jr.

4 A Stringent Yeast Two-Hybrid Matrix Screening Approach for Protein–Protein

Interaction Discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Josephine M. Worseck, Arndt Grossmann, Mareike Weimann, Anna Hegele,

and Ulrich Stelzl

5 High-Throughput Yeast Two-Hybrid Screening of Complex cDNA Libraries . . . . . 89

Kerstin Mohr and Manfred Koegl

6 Virus–Human Cell Interactomes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

Lionel Tafforeau, Chantal Rabourdin-Combe, and Vincent Lotteau

7 Interactome Mapping in Malaria Parasites: Challenges and Opportunities . . . . . . . . 121

Douglas J. LaCount

8 Mapping Interactomes with High Coverage and Efficiency Using

the Shifted Transversal Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147

Xiaofeng Xin, Charles Boone, and Nicolas Thierry-Mieg

9 Assigning Confidence Scores to Protein–Protein Interactions . . . . . . . . . . . . . . . . . 161

Jingkai Yu, Thilakam Murali, and Russell L. Finley Jr.

10 The Integration and Annotation of the Human Interactome

in the UniHI Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175

Gautam Chaurasia and Matthias Futschik

11 Gene-Centered Yeast One-Hybrid Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189

John S. Reece-Hoyes and Albertha J.M. Walhout

12 One- Plus Two-Hybrid System for the Efficient Selection of Missense

Mutant Alleles Defective in Protein–Protein Interactions. . . . . . . . . . . . . . . . . . . . . 209

Ji Young Kim, Ok Gu Park, and Young Chul Lee

13 Investigation of Membrane Protein Interactions Using the Split-Ubiquitin

Membrane Yeast Two-Hybrid System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225

Julia Petschnigg, Victoria Wong, Jamie Snider, and Igor Stagljar

viii Contents

14 Application of the Split-Protein Sensor Trp1 to Protein Interaction Discovery

in the Yeast Saccharomyces cerevisiae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245

Mandana Rezwan, Nicolas Lentze, Lukas Baumann, and Daniel Auerbach

15 Tetracycline Repressor-Based Mammalian Two-Hybrid Systems . . . . . . . . . . . . . . . 259

Kathryn Moncivais and Zhiwen Jonathan Zhang

16 The Fluorescent Two-Hybrid (F2H) Assay for Direct Analysis of Protein–Protein

Interactions in Living Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275

Kourosh Zolghadr, Ulrich Rothbauer, and Heinrich Leonhardt

17 ArrayMAPPIT: A Screening Platform for Human Protein Interactome Analysis. . . . 283

Sam Lievens, Nele Vanderroost, Dieter Defever, José Van der Heyden,

and Jan Tavernier

18 MAPPIT as a High-Throughput Screening Assay for Modulators

of Protein–Protein Interactions in HIV and HCV . . . . . . . . . . . . . . . . . . . . . . . . . . 295

Bertrand Van Schoubroeck, Koen Van Acker, Géry Dams, Dirk Jochmans,

Reginald Clayton, Jan Martin Berke, Sam Lievens, José Van der Heyden,

and Jan Tavernier

19 Integrated Measurement of Split TEV and Cis-Regulatory Assays Using

EXT Encoded Reporter Libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309

Anna Botvinik and Moritz J. Rossner

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325

ix

Contributors

DANIEL AUERBACH • Dualsystems Biotech Inc , Zurich , Switzerland

LUKAS BAUMANN • Dualsystems Biotech Inc , Zurich , Switzerland

JAN MARTIN BERKE • Tibotec Inc , Mechelen , Belgium

CHARLES BOONE • Terrence Donnelly Centre for Cellular and Biomolecular Research,

University of Toronto , Toronto , ON , Canada

ANNA BOTVINIK • Research Group ‘Gene Expression’ Max-Planck-Institute

of Experimental Medicine , Göttingen , Germany

GAUTAM CHAURASIA • Charité, Humboldt University , Berlin , Germany

REGINALD CLAYTON • Tibotec Inc , Mechelen , Belgium

GÉRY DAMS • Tibotec Inc , Mechelen , Belgium

DIETER DEFEVER • Department of Medical Protein Research,

VIB and Department of Biochemistry , Ghent University , Ghent , Belgium

RUSSELL L. FINLEY JR. • Center for Molecular Medicine and Genetics, Wayne State

University School of Medicine , Detroit , MI , USA

MATTHIAS FUTSCHIK • Centre for Molecular and Structural Biomedicine,

University of Algarve , Faro , Portugal

ARNDT GROSSMANN • Max Planck Institute for Molecular Genetics (MPI-MG) ,

Berlin , Germany

ROMAN HÄUSER • Karlsruhe Institute of Technology , Karlsruhe , Germany

ANNA HEGELE • Max Planck Institute for Molecular Genetics (MPI-MG) ,

Berlin , Germany

DIRK JOCHMANS • Tibotec Inc , Mechelen , Belgium

JI YOUNG KIM • School of Biological Sciences and Technology , Chonnam National

University , Gwangju , Republic of Korea

MANFRED KOEGL • Genomics and Proteomics Core Facility German Cancer

Research Institute , Heidelberg , Germany

DOUGLAS J. LACOUNT • Department of Medicinal Chemistry and Molecular

Pharmacology , Purdue University , West Lafayette , IN , USA

YOUNG CHUL LEE • School of Biological Sciences and Technology,

Chonnam National University , Gwangju , Republic of Korea

NICOLAS LENTZE • Dualsystems Biotech Inc , Zurich , Switzerland

HEINRICH LEONHARDT • Center for Integrated Protein Science (CiPSM)

and Department of Biology , Ludwig Maximilians University Munich ,

Planegg-Martinsried , Germany

SAM LIEVENS • Department of Medical Protein Research, VIB and Department

of Biochemistry , Ghent University , Ghent , Belgium

VINCENT LOTTEAU • Université de Lyon , Lyon , France

BERNARDO A. MANGIOLA • Center for Molecular Medicine and Genetics,

Wayne State University School of Medicine , Detroit , MI , USA

x Contributors

KERSTIN MOHR • Genomics and Proteomics Core Facility, German Cancer

Research Institute , Heidelberg , Germany

KATHRYN MONCIVAIS • College of Pharmacy, University of Texas at Austin ,

Austin , TX , USA

THILAKAM MURALI • Center for Molecular Medicine and Genetics, Wayne State

University School of Medicine , Detroit , MI , USA

OK GU PARK • School of Biological Sciences and Technology, Chonnam National

University , Gwangju , Republic of Korea

JODI R. PARRISH • Center for Molecular Medicine and Genetics, Wayne State

University School of Medicine , Detroit , MI , USA

JULIA PETSCHNIGG • Terrence Donnelly Centre for Cellular and Biomolecular

Research (CCBR), University of Toronto , Toronto , ON , Canada

CHANTAL RABOURDIN-COMBE • Université de Lyon , Lyon , France

SEESANDRA V. RAJAGOPALA • J Craig Venter Institute (JCVI) , Rockville , MD , USA

JOHN S. REECE-HOYES • University of Massachusetts Medical School ,

Worcester , MA , USA

MANDANA REZWAN • Dualsystems Biotech Inc , Zurich , Switzerland

GEORGE G. ROBERTS III • Center for Molecular Medicine and Genetics,

Wayne State University School of Medicine , Detroit , MI , USA

MORITZ J. ROSSNER • Research Group ‘Gene Expression’ Max-Planck-Institute

of Experimental Medicine , Göttingen , Germany

ULRICH ROTHBAUER • Center for Integrated Protein Science (CiPSM)

and Department of Biology , Ludwig Maximilians University Munich ,

Planegg-Martinsried , Germany

JAMIE SNIDER • Terrence Donnelly Centre for Cellular and Biomolecular

Research (CCBR), University of Toronto , Toronto , ON , Canada

IGOR STAGLJAR • Terrence Donnelly Centre for Cellular and Biomolecular

Research (CCBR), University of Toronto , Toronto , ON , Canada

THORSTEN STELLBERGER • Karlsruhe Institute of Technology , Karlsruhe , Germany

ULRICH STELZL • Max Planck Institute for Molecular Genetics (MPI-MG) ,

Berlin , Germany

LIONEL TAFFOREAU • Institute de biologie et de médecine moléculaires (IBMM),

Université libre de Bruxelles (ULB) , Gosselies , Belgium

JAN TAVERNIER • Department of Medical Protein Research, VIB and Department

of Biochemistry , Ghent University , Ghent , Belgium

NICOLAS THIERRY-MIEG • Laboratoire Techniques de l’Ingénierie Médicale

et de la Complexité - Informatique, Mathématiques et Applications de Grenoble

(TIMC-IMAG), Faculte de Medecine , La Tronche , France

PETER UETZ • Center for the Study of Biological Complexity Virginia

Commonwealth University , Richmond , VA , USA

KOEN VAN ACKER • Tibotec Inc , Mechelen , Belgium

JOSÉ VAN DER HEYDEN • Department of Medical Protein Research,

VIB and Department of Biochemistry , Ghent University , Ghent , Belgium

NELE VANDERROOST • Department of Medical Protein Research,

VIB and Department of Biochemistry , Ghent University , Ghent , Belgium

Contributors xi

BERTRAND VAN SCHOUBROECK • Tibotec Inc , Mechelen , Belgium

ALBERTHA J.M. WALHOUT • University of Massachusetts Medical School ,

Worcester , MA , USA

MAREIKE WEIMANN • Max Planck Institute for Molecular Genetics (MPI-MG) ,

Berlin , Germany

VICTORIA WONG • Terrence Donnelly Centre for Cellular and Biomolecular

Research (CCBR), University of Toronto , Toronto , ON , Canada

JOSEPHINE M. WORSECK • Max Planck Institute for Molecular Genetics (MPI-MG) ,

Berlin , Germany

XIAOFENG XIN • Terrence Donnelly Centre for Cellular and Biomolecular Research,

University of Toronto , Toronto , ON , Canada

JINGKAI YU • National Key Laboratory of Biochemical Engineering ,

Chinese Academy of Sciences , Beijing , China

ZHIWEN JONATHAN ZHANG • Bioengineering Program, School of Engineering,

Santa Clara University , Santa Clara , USA

KOUROSH ZOLGHADR • Center for Integrated Protein Science (CiPSM)

and Department of Biology , Ludwig Maximilians University Munich ,

Planegg-Martinsried , Germany

1

Bernhard Suter and Erich E. Wanker (eds.), Two Hybrid Technologies: Methods and Protocols, Methods in Molecular Biology,

vol. 812, DOI 10.1007/978-1-61779-455-1_1, © Springer Science+Business Media, LLC 2012

Chapter 1

Matrix-Based Yeast Two-Hybrid Screen Strategies

and Comparison of Systems

Roman Häuser , Thorsten Stellberger ,

Seesandra V. Rajagopala , and Peter Uetz

Abstract

Today, matrix-based screens are used primarily for smaller and medium-size clone collections in combination

with automation and cloning techniques that allow for reliable and fast interaction screening. Matrix-based

yeast two-hybrid screens are an alternative to library-based screens. However, intermediary forms are possible

too and we compare both strategies, including a detailed discussion of matrix-based screens. Recent

improvement of matrix screens (also called array screens) uses various pooling strategies as well as novel

vectors that increase their effi ciency while decreasing false-negative rates and increasing reliability.

Key words: Protein–protein interactions , Pooling , Mating , PI-deconvolution , Smart pool array

system , Shifted transversal design

Abbreviations

3-AT 3-Amino-1,2,4-triazole

AD Activation domain

DBD DNA-binding domain

GFP Green fl uorescent protein

GO Gene ontology

ORF Open reading frame

STD Shifted transversal design

Y2H Yeast two hybrid

Shortly after Stanley Fields and Ok-kyu Song invented the yeast

two-hybrid (Y2H) system in 1989 ( 1) , it was adapted for screens

of random libraries. Like the original Y2H assay, matrix-based

screens are usually carried out in living yeast cells although in the￾ory any other cell could be used. This is a crucial advantage since it

1. Introduction:

The Yeast Two￾Hybrid Principle

and Variations of It

2 R. Häuser et al.

represents an “in vivo” situation. The proteins of interest are provided

as plasmid-encoded recombinant fusion proteins (Fig. 1). The bait

protein is often fused to a DNA-binding domain (DBD) of the

yeast GAL4 transcription factor. The prey protein is tagged by

the activation domain (AD) of GAL4. A physical contact of the

bait and prey protein simulates the reconstitution of the GAL4

transcription factor. Once the bait protein is bound to its promoter

sequence by its DBD, the interacting proteins recruit the basal

yeast transcription machinery and thus activate the expression of a

reporter gene. Note that other fusion proteins can be used too and

have been established in other systems. For example, instead of the

Gal4 components, the bacterial transcription factor LexA has been

used. In general, any protein that can be split and reconstituted to

form an active protein can be used ( 2) .

For high-throughput screens, we routinely use the HIS3

auxotrophy marker. It encodes the essential enzyme imidazole￾glycerol-phosphate dehydratase which catalyzes the sixth step of

histidine biosynthesis. Hence, yeast growth on minimal medium

that lacks histidine can be used to indicate an interacting protein pair.

a

b

Y AD

HIS3 x

a

X Z

...

AD

HIS3 DBD DBD

X Y AD

HIS3

X Z AD

aa

a a

aa

bait prey library

diploid library

DBD

Fig. 1. The yeast two-hybrid principle. ( a ) Haploid yeast cells of mating type a are transfected

with a bait plasmid and those of mating type a with prey plasmids. A single bait strain is

mated with a prey library. ( b ) Resulting diploids ( a/ a ) carry the genetic material of mated

haploids. Interacting fusion proteins activate expression of the HIS3 reporter gene which

assures survival on minimal medium that lacks histidine (diploid on the left); diploids with

noninteracting fusions cannot grow (diploid on the right).