AN EXPRESSION CLONING METHOD TO IDENTIFY MONOMERIC GTP-BINDING PROTEINS BY GTP OVERLAY

We have developed a method for identifying monomeric GTP-binding prote ins that is based on probing plasmid expression libraries with [alpha- P-32]GTP. The method involves the production of nitrocellulose replica filter lifts from a plasmid cDNA expression library and treatment of the filters with chloroform vapor to lyse the Escherichia coli and to denature and inactivate endogenous E. coli GTP-binding proteins, thus allowing the direct identification of cDNA clones which encode Ras-lik e small GTP-binding proteins by ligand blotting. Using this procedure we have cloned a series of small Ras-like GTP-binding proteins from hu man retina. The method relies on a functional test, ligand specificity of the expressed proteins, to identify candidate molecules. This resu lts in the isolation of predominantly full-length cDNA clones without relying on DNA sequence similarity. Thus, this method may be particula rly useful for the cloning of novel Ras-related GTP-binding proteins w hich share limited sequence similarity with previously identified memb ers of the Ras superfamily. (C) 1997 Academic Press.