Rapid screening for protein interactors using in vitro translated protein and an expression library

S Robert, Robert Saint

Research output: Contribution to journalArticlepeer-review

Abstract

The identification of protein-binding partners for a particular protein of interest is a key strategy in defining the mechanism of action of that protein. The yeast two-hybrid screen (Ref. 1) is a powerful and simple method that has allowed many laboratories, including those without protein chemistry expertise, to identify protein interactors (reviewed in Ref. 2). The assay for interaction, in the case of the two-hybrid system, is transcriptional activation triggered as a result of protein interaction near the site of transcriptional initiation of a reporter gene. A potential limitation of this technique arises, therefore, if the protein used as the 'bait' acts as an activator or repressor in yeast leading, respectively, to activation or repression of the reporter gene regardless of any protein interactions that may occur. As alternatives, biochemical methods such as co-immunoprecipitation are technically challenging for those without protein chemistry expertise and identify the protein partner but do not provide its cDNA clone. We have developed a simple and rapid system for detecting protein binding partners that does not rely on a transcriptional assay but still provides a cDNA clone encoding the interacting protein. Although this method is based on an in vitro assay and is similar in principle to methods previously published (Ref. 3, 4), it has been designed so that no expertise with protein chemistry is required to use it successfully. The method is as simple as screening a bacteriophage DNA library with a radiolabelled DNA probe.
Original languageEnglish
JournalTechnical Tips Online
VolumeT01334
DOIs
Publication statusPublished - 1998

Fingerprint Dive into the research topics of 'Rapid screening for protein interactors using in vitro translated protein and an expression library'. Together they form a unique fingerprint.

Cite this