An expression cloning method to identify monomeric GTP-binding proteins by GTP overlay

Analytical Biochemistry
K Kadono-Okuda, D A Andres

Abstract

We have developed a method for identifying monomeric GTP-binding proteins that is based on probing plasmid expression libraries with [alpha-32P]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-like small GTP-binding proteins by ligand blotting. Using this procedure we have cloned a series of small Ras-like GTP-binding proteins from human retina. The method relies on a functional test, ligand specificity of the expressed proteins, to identify candidate molecules. This results in the isolation of predominantly full-length cDNA clones without relying on DNA sequence similarity. Thus, this method may be particularly useful for the cloning of novel Ras-related GTP-binding proteins which share limited sequence similarity with previously identified members of the Ras superfamily.

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Citations

Jan 20, 2010·Protein Engineering, Design & Selection : PEDS·Iain S MacPhersonMichael E P Murphy
Jan 25, 2005·Molecular and Biochemical Parasitology·Markus SpiliotisKlaus Brehm

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