Cloning to Identify Interacting Proteins

An important step in the characterization of any protein is to determine whether it exists in a complex with other proteins and, if it does, to identify its partners. This chapter describes a new technique to identify interacting proteins. It is a based on in vitro expression cloning (IVEC), which was recently developed to screen cDNA libraries rapidly and systematically according to the function of the protein encoded on each gene. This variation on traditional expression cloning has been successfully used to identify the substrates of kinases (Stukenberg et al. 1997), substrates of proteases (Cryns et al. 1997; Kothakota et al. 1997; Li 1998; McGarry and Kirschner 1998), DNA-binding activities (Mead et al. 1998), and components of signaling pathways (Andresson and Ruderman 1998). To date, IVEC has not been used to identify proteins that interact in complexes. This chapter describes a modification of the technique that successfully identified five binding partners of the specific phosphoserine/phosphothreonine-binding protein 14-3-3 (Kanai et al. 2000). Thus, IVEC can also complement the two-hybrid technique and biochemical purification to identify interacting proteins systematically. IVEC was originally reported in 1997 (Lustig et al. 1997; Stukenberg et al. 1997). The technique combines the power of expression cloning with the ability to manipulate a reaction exper-