An expression cloning method to identify monomeric GTP-binding proteins by GTP overlay.
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.
Kadono-Okuda
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Department of Biochemistry, University of Kentucky College of Medicine, Lexington 40536-0084, USA.
Andres
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DA
D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D019204:GTP-Binding Proteins / Q000302:isolation & purification*; D015723:Gene Library; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D015689:Oncogene Protein p21(ras) / Q000096:biosynthesis; D010957:Plasmids; D011197:Potassium Radioisotopes / Q000378:metabolism; D011869:Radioligand Assay; D017422:Sequence Analysis, DNA; D014162:Transfection; D018631:ras Proteins / Q000302:isolation & purification*
D018076:DNA, Complementary; D011197:Potassium Radioisotopes; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D015689:Oncogene Protein p21(ras); D018631:ras Proteins