UNIT 19.3 Phage-Based Expression Cloning to Identify Interacting Proteins
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
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. 15:19.3:19.3.1–19.3.9.
- Published Online: 1 MAY 2001
- Published Print: MAR 1999
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.