A fusion protein library: An improved method for rapid screening and characterization of DNA binding or interacting proteins

Masato Ikeda, Ken Ichi Arai, Hisao Masai*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

Abstract

A rapid method for screening and characterization of DNA binding or protein-interacting molecules is described. The method relies on a fusion protein library in which randomized DNA fragments are inserted into pGEX-3X and its derivatives to generate collections of GST-fusion proteins. After inducing the expression of the fusion proteins by addition of IPTG, the colonies can be screened either with radioactively labeled DNA/RNA fragment for specific clones encoding DNA/RNA binding proteins or with an antibody for clones encoding proteins of interest. They can also be screened with a radioactively labeled protein for cloning of interacting molecules. The fusion proteins encoded by the isolated clones can be readily purified by conducting the lysis of the cells and an affinity column in the presence of an alkyl anionic detergent, N-laurylsarcosine (sarkosyl), and can be further characterized.

Original languageEnglish (US)
Pages (from-to)167-171
Number of pages5
JournalGene
Volume181
Issue number1-2
DOIs
StatePublished - Nov 28 1996

Keywords

  • Affinity purification
  • Gene cloning
  • Glutathione S-transferase
  • South-western
  • West-western

ASJC Scopus subject areas

  • Genetics

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