Correlation of two-hybrid affinity data with in vitro measurements
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| Published in | Molecular and Cellular Biology Vol. 15; no. 10; pp. 5820 - 5829 |
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| Main Authors | , , |
| Format | Journal Article |
| Language | English |
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United States
American Society for Microbiology
01.10.1995
Taylor & Francis |
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| Online Access | Get full text |
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| AbstractList | Since their introduction, the interaction trap and other two-hybrid systems have been used to study protein-protein interactions. Despite their general use, little is known about the extent to which the degree of protein interaction determined by two-hybrid approaches parallels the degree of interaction determined by biochemical techniques. In this study, we used a set of lexAop-LEU2 and lexAop-lacZ reporters to calibrate the interaction trap. For the calibration, we used two sets of proteins, the Myc-Max-Mxi1 helix-loop-helix proteins, and wild-type and dimerization-defective versions of the lambda cI repressor. Our results indicate that the strength of interaction as predicted by the two-hybrid approach generally correlates with that determined in vitro, permitting discrimination of high-, intermediate-, and low-affinity interactions, but there was no single reporter for which the amount of gene expression linearly reflected affinity measured in vitro. However, some reporters showed thresholds and only responded to stronger interactions. Finally, some interactions were subject to directionality, and their apparent strength depended on the reporter used. Taken together, our results provide a cautionary framework for interpreting affinities from two-hybrid experiments.Since their introduction, the interaction trap and other two-hybrid systems have been used to study protein-protein interactions. Despite their general use, little is known about the extent to which the degree of protein interaction determined by two-hybrid approaches parallels the degree of interaction determined by biochemical techniques. In this study, we used a set of lexAop-LEU2 and lexAop-lacZ reporters to calibrate the interaction trap. For the calibration, we used two sets of proteins, the Myc-Max-Mxi1 helix-loop-helix proteins, and wild-type and dimerization-defective versions of the lambda cI repressor. Our results indicate that the strength of interaction as predicted by the two-hybrid approach generally correlates with that determined in vitro, permitting discrimination of high-, intermediate-, and low-affinity interactions, but there was no single reporter for which the amount of gene expression linearly reflected affinity measured in vitro. However, some reporters showed thresholds and only responded to stronger interactions. Finally, some interactions were subject to directionality, and their apparent strength depended on the reporter used. Taken together, our results provide a cautionary framework for interpreting affinities from two-hybrid experiments. Since their introduction, the interaction trap and other two-hybrid systems have been used to study protein-protein interactions. Despite their general use, little is known about the extent to which the degree of protein interaction determined by two-hybrid approaches parallels the degree of interaction determined by biochemical techniques. In this study, we used a set of lexAop-LEU2 and lexAop-lacZ reporters to calibrate the interaction trap. For the calibration, we used two sets of proteins, the Myc-Max-Mxi1 helix-loop-helix proteins, and wild-type and dimerization-defective versions of the lambda cI repressor. Our results indicate that the strength of interaction as predicted by the two-hybrid approach generally correlates with that determined in vitro, permitting discrimination of high-, intermediate-, and low-affinity interactions, but there was no single reporter for which the amount of gene expression linearly reflected affinity measured in vitro. However, some reporters showed thresholds and only responded to stronger interactions. Finally, some interactions were subject to directionality, and their apparent strength depended on the reporter used. Taken together, our results provide a cautionary framework for interpreting affinities from two-hybrid experiments. Article Usage Stats Services MCB Citing Articles Google Scholar PubMed Related Content Social Bookmarking CiteULike Delicious Digg Facebook Google+ Mendeley Reddit StumbleUpon Twitter current issue Spotlights in the Current Issue MCB About MCB Subscribers Authors Reviewers Advertisers Inquiries from the Press Permissions & Commercial Reprints ASM Journals Public Access Policy MCB RSS Feeds 1752 N Street N.W. • Washington DC 20036 202.737.3600 • 202.942.9355 fax • journals@asmusa.org Print ISSN: 0270-7306 Online ISSN: 1098-5549 Copyright © 2014 by the American Society for Microbiology. For an alternate route to MCB .asm.org, visit: MCB |
| Author | R Brent E A Golemis J Estojak |
| AuthorAffiliation | Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA |
| AuthorAffiliation_xml | – name: Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA |
| Author_xml | – sequence: 1 givenname: Joanne surname: Estojak fullname: Estojak, Joanne organization: Fox Chase Cancer Center – sequence: 2 givenname: Roger surname: Brent fullname: Brent, Roger organization: Department of Genetics, Harvard Medical School – sequence: 3 givenname: Erica A. surname: Golemis fullname: Golemis, Erica A. email: EA_Golemis@fccc.edu organization: Fox Chase Cancer Center |
| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/7565735$$D View this record in MEDLINE/PubMed |
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Mendeley... Since their introduction, the interaction trap and other two-hybrid systems have been used to study protein-protein interactions. Despite their general use,... |
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| SubjectTerms | Bacterial Proteins - genetics Base Sequence beta-Galactosidase - genetics DNA - metabolism DNA-Binding Proteins - metabolism Genes, Reporter - genetics Helix-Loop-Helix Motifs Kinetics Leucine Molecular Sequence Data Mutation Operator Regions, Genetic phage lambda Protein Binding Recombinant Fusion Proteins - biosynthesis Repressor Proteins - genetics Repressor Proteins - metabolism Saccharomyces cerevisiae - genetics Serine Endopeptidases Transcriptional Activation Viral Proteins Viral Regulatory and Accessory Proteins |
| Title | Correlation of two-hybrid affinity data with in vitro measurements |
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