Expression cloning to identify monomeric GTP-binding proteins by GTP overlay

Abstract

More than 70 small Ras-related GTP-binding proteins have been identified and additional members of the family continue to be discovered. The majority of these proteins have been isolated by screening genomic or cDNA libraries at low stringency with oligonucleotide mixes corresponding to the conserved guanine nucleotide-binding regions or to short peptide sequences conserved within individual Ras-related subfamilies. Although these strategies have proved successful, their use in the identification of novel mammalian small GTP-binding proteins, or of Ras-related proteins in plants and lower organisms, may not be as effective, particularly if this novel GTPase is distant in sequence from previously identified Ras family members. This chapter describes an expression cloning strategy that allows the rapid isolation of full-length cDNA encoding small GTP-binding proteins that may escape detection by conventional cloning methods. Identification of novel GTP-binding proteins as well as a complete cataloging of all Ras-related GTPase provide new insight into the diverse cellular pathways regulated by these proteins and are critical in understanding the regulation and complicated biological interactions among members of the Ras protein family. The approach relies on the proved ability of Ras-related proteins to bind GTP after denaturation and transfer to nitrocellulose membrane.