Tai Lieu Chat Luong METHODS IN 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 Editors Bernhard Suter Max-Delbrück-Centrum für Molekulare Medizin Quintara Biosciences Albany, CA, USA bernhard.suter@mdc-berlin.de Erich E Wanker Max-Delbrück-Centrum für Molekulare Medizin Berlin-Buch, Germany ewanker@mdc-berlin.de 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 publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights Printed on acid-free paper Humana Press is part of Springer Science+Business Media (www.springer.com) 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 field is largely driven by the development of innovative technologies and strategies for efficient 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 variants, 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 complementation and validation of Y2H screens The book is structured into two sections The first gives a survey of protocols that are currently employed for Y2H high-throughput screens by different expert labs in the field 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 highthroughput 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 interactomes), 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 confidence scoring, whereas Gautam Chaurasia and Matthias Futschik describe the design of a database for highthroughput Y2H data (UniHI, Max Delbrueck Centrum, Berlin) John Reece-Hoyes and Albertha Walhout present a high-throughput yeast one-hybrid variant for the identification of proteins that bind-specific DNA segments Finally, contributors from the lab of Young Chul Lee introduce their “one- plus two-hybrid system” for the efficient identification 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 v vi Preface Zhang A different principle in mammalian cells is used by Heinrich Leonhardt and team in their fluorescent two-hybrid approach, where bait and prey proteins are recruited to specific 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 interaction 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 profiling Together, the protocols supply researchers with a comprehensive toolbox for the identification 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 Berlin, Germany Bernhard Suter Erich E Wanker Contents Preface Contributors v ix Matrix-Based Yeast Two-Hybrid Screen Strategies and Comparison of Systems Roman Häuser, Thorsten Stellberger, Seesandra V Rajagopala, and Peter Uetz Array-Based Yeast Two-Hybrid Screens: A Practical Guide 21 Roman Häuser, Thorsten Stellberger, Seesandra V Rajagopala, and Peter Uetz High-Throughput Yeast Two-Hybrid Screening 39 George G Roberts III, Jodi R Parrish, Bernardo A Mangiola, and Russell L Finley Jr 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 High-Throughput Yeast Two-Hybrid Screening of Complex cDNA Libraries 89 Kerstin Mohr and Manfred Koegl Virus–Human Cell Interactomes 103 Lionel Tafforeau, Chantal Rabourdin-Combe, and Vincent Lotteau Interactome Mapping in Malaria Parasites: Challenges and Opportunities 121 Douglas J LaCount Mapping Interactomes with High Coverage and Efficiency Using the Shifted Transversal Design 147 Xiaofeng Xin, Charles Boone, and Nicolas Thierry-Mieg 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 vii 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 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, Gưttingen, 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 ix 19 Integrated Measurement of Split TEV and Cis-Regulatory Assays… 315 medium, 50 mg/ml kanamycin and are allowed to grow at 37°C for h with mild shaking After subcloning, the EXT library has to be screened for full length unique EXTs to exclude repeating sequences and products of incomplete oligonucleotide synthesis 10 Option 1: Analysis of individual entry clones A portion of the bacteria are kept at 4°C over-night and appropriate volumes are plated according to the determination of recombinants adjusted to 100–500 clones per 10 cm LB-agar dish 11 From these plates, e.g., 96 colonies are screened by “colony PCR” by dipping a sterile tooth pick into wells of a 96-well PCR plate containing all reagents including the primers pENTR_s and Dec2 Subsequently, the tooth pick is transferred to the corresponding positions in 96-deep well dishes (with ~1 ml LB/well) and grown at 37°C for at least 16 h The PCR products are separated on 4% agarose gels and clones are discarded that show reduced insert length ( features per EXT) and a set of hybridization specificity controls including EXT sequence permutations carrying mismatches at different sites along the sequence and varying numbers of mismatches to determine optimal hybridization stringency (see Botvinnik et al., 2010) 19 Integrated Measurement of Split TEV and Cis-Regulatory Assays… 323 References Papin JA, Hunter T, Palsson BO, Subramaniam S (2005) Reconstruction of cellular signalling networks and analysis of their properties Nat Rev Mol Cell Biol 6, 99–111 Citri A, Yarden Y (2006) EGF-ERBB signalling: towards the systems level Nat Rev Mol Cell Biol 7, 505–516 Jones RB, Gordus A, Krall JA, MacBeath G (2006) A quantitative protein interaction network for the ErbB receptors using protein microarrays Nature 439, 168–174 Schulze WX, Deng L, Mann M (2005) Phosphotyrosine interactome of the ErbBreceptor kinase family Mol Syst Biol 1, 2005 0008 Brenner S, Williams SR, Vermaas EH, Storck T, Moon K, McCollum C, et al (2000) In vitro cloning of complex mixtures of DNA on microbeads: physical separation of differentially expressed cDNAs Proc Natl Acad Sci USA 97, 1665–1670 Botvinnik A, Wichert SP, Fischer TM, Rossner MJ (2010) Integrated analysis of receptor activation and downstream signaling with EXTassays Nat Methods 7, 74–80 Wehr MC, Laage R, Bolz U, Fischer TM, Grunewald S, Scheek S, et al (2006) Monitoring regulated protein-protein interactions using split TEV Nat Methods 3, 985–993 Wehr MC, Reinecke L, Botvinnik A, Rossner MJ (2008) Analysis of transient phosphorylationdependent protein-protein interactions in living mammalian cells using split-TEV BMC Biotechnol 8, 55 sdfsdf INDEX A Activation 5, 8, 12, 25, 28, 31, 35, 40, 53, 57–59, 77, 90–92, 102, 108–109, 116, 122, 124, 142, 153, 177, 180, 182, 189, 190, 195, 203–206, 210, 211, 214, 217, 284, 285, 291, 309, 310, 316, 317 Activation domain (AD) 2, 7, 15–17, 22, 25, 33, 40–44, 47, 48, 50–59, 68, 102, 104, 106–109, 111, 112, 115, 117, 122–126, 128–136, 140–144, 150, 153, 154, 189–191, 194, 198–203, 205, 210, 211, 214, 217 library 40, 47, 48, 52, 57, 124, 125, 128, 129, 132, 136, 140, 141, 143, 144 ADE2 reporter gene 142 ADH1 promoter 42, 43, 47, 68, 69, 104, 105, 115, 116, 124, 241 Agar 5, 6, 25, 27–36, 42–44, 46, 48, 52–54, 56–58, 66–68, 71, 73–84, 90, 91, 94, 95, 97, 98, 101, 106, 127, 152, 155, 156, 158, 192, 193, 231, 248–250, 262, 311, 315, 316 3-Amino–1,2,4-triazole (3-AT) 25, 26, 29, 32, 33, 35, 37, 92, 94, 97–99, 107–110, 113, 114, 116, 128, 140–142, 150, 152, 156, 157, 228–232, 235, 236, 238, 242 Amplification 98–99, 111, 117, 128–129, 132, 134, 137, 201, 228, 234, 235, 241, 248, 251, 254, 255, 277, 318, 320, 322 Amyloid precursor protein (APP) 255 Antibody 229, 263, 264, 271–273 Arrays Array screens 14–16, 25, 28, 64, 111–114 Autoactivation .70, 71, 75–77, 79, 83, 195–196, 203, 204 Automated screening 78 Auxotrophy, marker 2, B Bacterial genome 7, 21, 27 Bacteriophage T7 3, 18 B42 activation domain 211, 214, 217 Bait 2, 16–17, 22, 25–26, 30–32, 40, 64, 77–78, 90–94, 96–100, 104, 106–118, 153, 191–207, 210, 211, 214–217, 220, 226–231, 233–238, 240–243, 247, 248, 250–252, 254–257, 260–265, 267, 268, 270, 272, 276, 284–286, 288–293, 296 Bait-dependency 229–230, 236, 238, 243, 247, 250, 255–257 Beta-galactosidase 3, 71, 80–82, 84, 91, 92 Binary interaction 52, 63, 284 Binding affinity 4, C cDNA library 47, 107, 109, 116, 117, 124, 129, 143, 190, 192, 194, 198–202, 204–206, 246, 247, 250, 252–253, 255, 257 Chaperones 8, 81 Chimeric receptor 296, 298 Classification 16, 64, 93, 157, 162, 172, 173, 240 Colony 3, 6, 8, 9, 27, 28, 35, 49–51, 57, 8, 64, 73, 79, 83, 90, 92, 93, 97, 98, 101, 111, 112, 114, 117, 134, 136, 139, 147, 156, 157, 197, 206, 219, 230, 235, 237, 239, 240, 250, 252, 266, 267, 315, 316 Colony forming unit (CFU) 49, 58, 110, 136, 140, 141, 144, 253 Complexity 40, 59, 89, 90, 99, 100, 176, 204, 205, 283, 313–317 Confidence score 164 Contamination 8, 36, 37, 45, 55, 59, 83, 101, 117, 136, 141, 195, 199, 322 Counter-selection 210 Coverage 40, 47, 104, 137, 139, 147–158 C-terminal fusion 16–17, 124 D Database .6, 52, 54–56, 79, 90, 92, 99, 102, 104, 114, 115, 165–167, 170–172, 175–184 Deconvolution 11–12, 77 Destination vector 23–25, 68, 69, 71, 82, 83, 105, 106, 116, 289, 311, 315 Dextrose (D) .42, 106, 127, 212 Diploid yeast 3, 54, 71, 77, 81, 140–142, 192 Dissociation 261, 285 DNA isolation 104, 311, 316–319 preparation 112, 219, 277, 279, 315, 317 solution (mix) 129, 214 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, © Springer Science+Business Media, LLC 2012 325 TWO HYBRID TECHNOLOGIES: METHODS AND PROTOCOLS 326 Index DNA binding domain (DBD) 2, 14–16, 22, 25, 33, 41, 68, 104, 122–124, 126–128, 136, 139–142, 144, 153, 207, 211, 214, 226, 260 Domain–domain interactions 165, 167 Donor vector 311 Drop-out medium 127, 212, 231 E Efavirenz (EFV) .296, 297, 299, 302–304 Effectene 287, 290, 293 Escherichia coli (E.coli) 14, 15, 26, 30, 41, 69–70, 72–74, 80, 83, 125, 129, 130, 142, 219, 222, 228–230, 234, 238–240, 242, 262, 266, 279 Expressed oligonucleotide tags (EXTs) 309–322 Expression 2, 3, 5, 8, 23, 24, 26, 35, 41–43, 64, 68–70, 89–91, 103, 108, 115, 116, 122, 123, 128, 137, 140, 142, 143, 162, 165–167, 169, 170, 172, 177, 179–182, 184, 191, 196, 200, 203, 205, 210–212, 215, 227, 240–242, 247, 248, 252, 260, 261, 263–264, 268, 270–272, 276, 277, 279, 281, 289, 298, 311–317, 320–322 F False negatives 6–9, 11, 12, 17, 27, 148, 156, 162 False positives 6–9, 11, 12, 28, 52, 55, 58, 115, 122, 123, 142, 148, 156, 157, 161–163, 183, 210, 219, 222, 223, 268, 281 Fluoresecent two-hybrid (F2H) 275–281 5-Fluoroorotic 210 Format 4, 7, 12, 22, 25, 27, 32, 41, 42, 46, 52, 64, 71, 72, 75, 77–79, 84, 111–115, 122, 144, 152–157, 195, 200, 202, 289 Forward primer 197, 212, 217, 234, 235, 254, 279 Full length protein 7, 22, 104 Fusion protein 2, 3, 7, 16, 24, 33, 57, 64, 77, 115, 116, 122, 123, 140, 141, 211, 212, 247, 248, 251, 257, 276, 280, 281, 296, 298 G Galactose 41–45, 48, 50, 80, 214, 215 GAL4 transcription factor 2, 3, 41, 104, 191 Gap-repair 106, 107, 112–114, 118, 124, 201–202, 205, 206, 217, 228, 231, 234 Gateway cloning 23–25, 69, 71–72, 191, 202, 289 Gene expression 3, 5, 41, 69, 165–167, 177, 181, 184, 205, 227, 260, 268 Gene ontology (GO) 8, 166, 170, 177, 184 Geneticin 232, 299 Genomics 3, 6, 41, 170, 178, 229 Glucose .28, 29, 41–45, 50, 67, 68, 70, 82, 127, 150–152, 192, 193, 205, 214, 215, 231, 249, 250 Glycerol 31, 34, 43–45, 53, 57, 58, 67, 70, 73, 110, 117, 125, 126, 150, 155, 158, 197, 221, 263, 267, 287, 289 Gold standard 15, 163, 183 Green fluorescent protein (GFP) 3, 69, 263, 268, 270, 271, 276, 277, 280 H Haploid yeast 2, 3, 25, 40, 104 HCV-human interaction network 104 Hek293 cells .262, 267, 268, 270 Herpesvirus Heterodimerization 261, 304 Heterologous 8, 241, 242 High-density 153, 157, 158 High-quality 64, 66, 129, 157, 288 High-throughput 2, 4, 21–24, 27, 39–59, 89–102, 147, 148, 162, 169, 171–173, 176, 183, 237–238, 261, 276, 278–280, 284, 295–306 HIS3 reporter gene 2, 3, 25, 92, 108, 116, 142, 210, 212, 214, 222 Histidine (H) 2, 5, 29, 42, 47, 67, 91, 107–109, 127, 151, 152, 192, 204, 210–212, 214, 231, 249, 250 Homodimerization 261 Hubs 148 I Image processing 156 Immunofluorescence 229, 235–237 Infection network 104 Inoculation 49, 73, 74 Interaction 3, 25, 39, 63–84, 103, 123, 148, 161–173, 175, 189, 209–223, 225–243, 245–257, 259, 275–281, 283, 295–306 Interaction-defective mutant allele 210, 218, 219 Interaction mapping 63, 65, 104, 168, 176–179, 183, 283 Interaction sequence tag (IST) 111, 119 Interactome .63, 103–118, 121–144, 147–158, 175–184, 283–293 mapping 63, 121–144, 147–158, 284 Interologs 16, 164–165, 169, 171, 172 In vivo method 2, 6, 21, 24, 112, 138, 161–163, 191, 206, 217, 226, 234, 259–261 J Janus kinase ( JAKs) 285, 288, 289, 296, 303, 304 K Kanamycin 124, 230, 231, 279, 311, 315 L LacZ reporter gene 116, 241 Large-scale 3, 4, 9, 17, 40, 63–65, 135–136, 158, 173, 176, 177, 189, 191, 257, 283, 284 TWO HYBRID TECHNOLOGIES: METHODS AND PROTOCOLS 327 Index Leucine 3, 29, 30, 41–43, 48, 58, 67, 70, 75, 107, 123, 127, 151, 152, 192, 226, 231, 250 LEU2 gene 3, 36, 41, 43, 47, 50, 54–56, 59, 68, 94, 106, 116, 124, 153, 193, 212, 214, 221, 230, 231, 248 LexA transcription factor 2, 227 Library screens 3, 6, 9, 14, 28, 40, 41, 47–52, 58, 64, 93, 107–111, 122, 124, 128, 190, 197, 203–205, 250–254 Ligation 23–25, 130, 138, 234, 265, 266, 277, 279 Lithium acetate (LiOAc) 29, 31, 70, 76, 128, 133, 135, 144, 193, 229, 232, 237, 249, 251–253 Luria Bertani (LB) 67, 70, 73, 83, 219, 230, 231, 239, 262, 266, 286, 289, 292, 311, 314–316 M Mammalian protein–protein interaction trap (MAPPIT) .65, 283–293, 295–306 Mammalian two-hybrid 259–273 Mating efficiency 6, 7, 27, 36, 83 Mating type 2–4, 6, 25, 40, 70, 91, 110 Matrix 1–18, 27, 45, 63–84, 111, 131, 152, 155 Matrix-based screens 1–18 Membrane protein 22, 183, 225–243, 246, 255 Membrane yeast two-hybrid 225–243 Microarray 11, 181, 191, 310, 312, 319–322 Micro-pool 12, 13, 148–150, 152–155, 158 Mini-pool 9, 10, 33–34 Missense mutations 209–223 Multiplexing .310, 316, 317, 322 Mutagenic PCR 212, 217, 218 N Negative set 165, 168 Neomycin 262, 298 Network analysis 176, 181–182 biology 63 neighbors 168 Neuregulin-ErB 309 Non-interactor 58, 172, 210, 222 N-terminal fusion 16–17 Nucleus 7, 162, 226, 227, 276, 277, 281, 285, 296 O Omnitrays 7, 28–30, 33, 44–46, 48, 50, 51, 66, 76, 144, 152 One-on-one array 12, 149, 151 One-plus two-hybrid system 209–223 Open reading frame (ORF) .6, 7, 14–17, 22–25, 27, 40, 41, 66, 68, 71, 82, 89, 93, 99, 104, 114, 115, 122, 149, 153, 171, 192, 205, 289 ORFeome 22–25, 114, 115, 149, 150, 153, 154, 286, 288 library 7, 99 Orthologs 22, 124, 165, 166, 171, 172, 177, 178, 180, 183 Overexpression .8, 227, 240, 242 P PCR See Polymerase chain reaction (PCR) Peptone .28, 43, 45, 67, 82, 106, 127, 152, 192, 212, 231, 248 Plasmid 2, 23–24, 40, 47, 69, 91, 93, 96, 100, 104–106, 108, 122, 136–140, 153, 189, 212, 214, 227, 228, 247–248, 250–251, 261–262, 264–267, 276, 284, 288–290, 298, 311 Plasmodium falciparum 121–124, 126, 128–129, 136–140 Polyethylene glycol (PEG) 29, 70, 76, 91, 94, 95, 97, 100, 107–109, 113, 116, 118, 128, 131, 135, 144, 193, 195, 198, 199, 201, 229, 232, 237, 249, 251–253, 256, 315 Polymerase chain reaction (PCR) 6, 9, 22–24, 31, 45, 46, 50–52, 66, 71–73, 75, 76, 90, 95–96, 98–102, 111–113, 117, 118, 123, 125–139, 141–144, 193–194, 196–199, 201, 202, 204–206, 212, 214, 217, 218, 222, 228, 230, 234, 235, 240, 241, 250, 251, 254–256, 261, 262, 264, 265, 277, 279, 292, 311–316, 318–320, 322 product 9, 23, 24, 31, 51, 52, 90, 92, 102, 112, 113, 118, 123, 131, 133–139, 141–144, 197, 201, 202, 206, 217, 218, 234, 241, 255, 265, 313, 315, 316, 319 Pooling .6, 7, 9–13, 27, 33–34, 40, 52, 77, 78, 110, 147–150, 154, 158, 317 Pool size 52, 77, 148, 150, 153, 154, 157 Positive set 165 Prey 2–17, 22–36, 58, 59, 64, 68–73, 75–81, 83, 84, 90–95, 97–99, 102, 104–114, 116–118, 147–150, 153–156, 201–203, 205, 206, 210, 211, 214–219, 221, 222, 226–229, 235–238, 242, 243, 247, 248, 254, 255, 260–267, 270, 272, 276–279, 281, 284–293, 296, 298, 300 Protein 2, 21, 40, 63–85, 90, 92, 99, 102, 103, 121, 147, 161–173, 175, 189, 209–223, 225–243, 245–257, 259, 275–281, 283–293, 295–306, 309 complex 4, 27, 167, 211, 221, 245 folding 4, fragment 7, 66, 284, 285 interaction database 165 processing 7, 297 topology 226 Protein complementation assay (PCA) 284 TWO HYBRID TECHNOLOGIES: METHODS AND PROTOCOLS 328 Index Protein–protein interaction 63–85, 103, 104, 123, 124, 148, 161–173, 176, 177, 182, 189, 209–223, 225, 226, 245, 255, 259–261, 275–281, 283, 284, 289, 295–306, 316, 317 Proteome 3, 9, 13, 21, 40, 52, 63, 103, 113, 165, 184, 226, 284 R Reconstitution 2, 112, 226, 247 Redundancy 5, 11–13, 148–150, 154, 166 Replica plating .46, 143, 153, 156, 193, 200, 201, 203 Reproducibility 4, 5, 8, 14, 34, 54, 157 Retest 8–11, 27, 28, 34–35, 37, 52, 55, 64, 65, 71, 75, 77–81, 84, 107, 112–113, 124, 141, 142, 152, 153, 156–157, 190, 201–203, 205, 206, 219, 292 Reverse primer 212, 217, 234, 235, 254, 262, 276, 279 Reverse transfection 284, 286, 288–293 Reverse two-hybrid system 210, 223 Robotics 7, 21, 22, 26, 36, 45, 53, 92, 111, 118, 152, 156, 157, 286, 288, 292 S Saccharomyces cerevisiae 4, 69, 124, 171, 176, 230, 245–257 Salmon sperm DNA 29, 71, 95, 128, 193, 194, 198, 232, 249, 256 Scoring .22, 33, 58, 59, 112, 113, 151, 156, 158, 161–164, 168–170, 172, 173 Screening 4, 6, 9–12, 14, 22–27, 29, 32, 35, 39–59, 63–85, 89–102, 104, 105, 107–114, 122, 127–128, 140, 147–150, 153, 155–158, 192, 194, 198–202, 210, 211, 213–219, 221, 222, 233, 235, 237–238, 240–242, 246, 247, 250–254, 261, 268, 275, 280, 283–293, 295–306, 315 cost 149 efficiency 12 method 40–41, 112 protocol 35, 71, 89, 153, 300–302 Screens 1–9, 11, 12, 14–17, 21–37, 40, 41, 47, 58, 64, 65, 77, 79, 84, 90, 92–95, 97–101, 104, 115, 122, 124, 128, 136, 140–141, 152, 155, 156, 162, 169, 172, 173, 177, 180, 197, 203–205, 207, 226, 231, 241, 278, 279 Second generation DNA sequencing 148 Self-activation 5, 8, 12, 25–26, 30–33, 35, 107–110, 116–118, 124, 140, 144, 236 Sensitivity 7–9, 12, 27, 36, 40, 41, 65, 77, 149, 158, 183, 257, 259, 260 Sensor 245–257 Sequencing 6, 7, 9, 40, 41, 51, 52, 73, 90, 95, 99, 101, 102, 111, 115, 141, 142, 147, 148, 191, 202, 206, 219, 228, 229, 234, 235, 238, 241, 242, 247, 248, 250, 251, 255, 262, 267, 279, 310, 315 Shifted transversal design (STD) 12, 13, 147–158 Signal-to-noise ratio 5, 281 Signal transduction 175, 303 Small-scale 22, 175, 217, 218 Smart pool array (SPA) system 12 Specificity 8, 9, 12, 77, 124, 149, 158, 183, 302–303 Split-protein 245–257 Split-TEV 309–322 Split-ubiquitin 3, 225–243 Stanley Fields Sticky protein Stringency 64, 92, 157, 236, 242, 322 Synthetic complete medium 192 Systems biology 192 T Tetracycline operator 260 repressor 259–273 Therapeutic targets 175, 296 Thyroid hormone receptor-associated protein 220 212 Tissue culture 262 Toxicity 94, 303 Training set 8, 28, 163, 164, 168–170, 172 Transcription 2, 3, 14, 24, 25, 41, 68, 92, 104, 175, 184, 189–191, 221, 226, 227, 246, 260, 284, 285, 296, 311, 313, 317, 320 factor 2, 3, 41, 104, 190, 191, 226, 227, 246, 285 Transformation 29–31, 69–73, 75, 76, 83, 95–97, 105, 107–109, 112, 113, 116–118, 127, 128, 133–136, 138, 139, 142, 143, 193–195, 198, 199, 201–203, 205, 206, 215, 218, 219, 222, 228–230, 234–239, 242, 244, 248, 250–253, 255, 256, 266, 277, 279 Treponema pallidum 4, 14 TRP1 gene 246 True negatives 161 True positives 59, 123, 157, 162, 163, 168, 170 Tryptophan 29, 30, 42, 47, 67, 70, 75, 107, 109, 115, 123, 127, 151, 152, 192, 204, 231, 246, 247, 249, 250, 255, 256 Two-phase strategy 10 U Ubiquitin 3, 81, 225–243, 246, 284 Unified Human Interactome (UniHI) database 175–184 Univector 22, 23 Uni vector-Plasmid-Fusion system 23 Uracil 3, 29, 42, 47, 67, 122, 123, 127, 151, 192, 204, 214, 231, 296 URA4 gene 123 URA3 reporter gene 94, 210 TWO HYBRID TECHNOLOGIES: METHODS AND PROTOCOLS 329 Index V Y Validation 16, 41, 162, 221, 228, 233, 236, 240, 284, 302 Vector 7, 14–16, 22, 40, 66, 68–69, 95, 104, 122, 199, 210, 213–217, 228, 247, 261, 276, 285, 298, 311 Virus–host interactomes 103 Virus–human interaction 103 Vitamin D receptor 209, 212 VP16 interaction domain 210, 226, 227, 229, 236, 241 Yeast 1, 21, 39, 64, 89–102, 104, 122, 151, 162, 189, 210, 227, 245–256, 260, 286 extract 28, 43, 45, 67, 70, 82, 106, 127, 152, 192, 212, 222, 231, 248, 286, 287 one-hybrid 189–207 two-hybrid .1–17, 21–37, 39–59, 63–84, 89–102, 104, 106–109, 111–112, 121–124, 127–129, 136, 140–142, 144, 147, 153, 161, 162, 177, 189, 209, 210, 220, 225–243, 246, 260, 284 X X-gal plates .50, 210–212, 219, 222