Unit

UNIT 19.3 Phage-Based Expression Cloning to Identify Interacting Proteins

  1. Julie M. Stone

Published Online: 1 MAY 2001

DOI: 10.1002/0471140864.ps1903s15

Current Protocols in Protein Science

Current Protocols in Protein Science

How to Cite

Stone, J. M. 2001. Phage-Based Expression Cloning to Identify Interacting Proteins. Current Protocols in Protein Science. 15:19.3.1–19.3.9.

Author Information

  1. Massachusetts General Hospital, Boston, Massachusetts

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: MAR 1999

Abstract

Interaction cloning (also known as expression cloning) is a technique to identify and clone genes which encode proteins that interact with a protein of interest, or “bait” protein. The procedure presented in this unit involves a fusion protein consisting of bait protein and glutathione-S-transferase (GST) with a protein kinase A site at the junction between them (the protocol can, however, be adapted to use other PKA-containing recombinant proteins). The labeled protein is subsequently used as a probe to screen a l bacteriophage-derived cDNA expression library, which expresses b -galactosidase fusion proteins that contain in-frame gene fusions. The phages lyse cells, form plaques, and release fusion proteins that are adsorbed onto nitrocellulose membrane filters. The filters are blocked with excess nonspecific protein to eliminate nonspecific binding and probed with the radiolabeled bait protein. This procedure leads directly to the isolation of genes encoding the interacting protein, bypassing the need for purification and microsequencing or for antibody production.