We have developed a method for identifying monomeric GTP-binding proteins that is based on probing plasmid expression libraries with [α- 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.
|Number of pages||5|
|State||Published - Dec 15 1997|
Bibliographical noteFunding Information:
1This work was supported by National Institutes of Health Grant EY 11231.
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology