Protein Regulates Cell Proliferation Research Articles

15] Identification of protein-protein interactions by λgt11 expression cloning

This chapter discusses the identification of protein–protein interactions by λgt11 expression cloning. Protein–protein interactions govern a wide variety of fundamental biological processes, ranging from the control of enzymatic activity through subunit association and enzyme-substrate recognition, to the specific interactions of protein ligands with their receptors. Intermolecular associations are also central to the manner in which oncogene-encoded proteins regulate cell proliferation. The realization that highly specific protein–protein interactions mediate cell behavior at almost every level has led to attempts to develop general methods with which to identify molecules that physically associate with a given protein. These approaches fall roughly into two classes: biochemical and genetic. When correct processing and post-translational modification are a prerequisite for molecular interaction, a modified genetic strategy that employs transfection of a cDNA expression library into mammalian cells can be used. Such a technique has been used quite successfully to identify several cell surface receptors based on their ligand binding activities. A frequently used biochemical approach to identify specific molecular interactions has been to employ an immobilized target protein to adsorb unknown binding partners from cell extracts. One advantage of the expression cloning method is that the actual screening is carried out ex vivo. This permits a greater degree of control over the preparation and labeling of the probe than in the two-hybrid system.