A cDNA cloning method for monomeric GTP-binding proteins by ligand blotting.
We have developed a convenient method of cDNA cloning for monomeric GTP-binding proteins. The method consists of a serial dilution of cDNA expression library, a separation of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and a ligand blotting; each group containing the proteins from about 1000 independent colonies was separated by SDS-PAGE, transferred to nitrocellulose filters, incubated with 10(-9) M [alpha-32P]GTP, and autoradiographed. Separation of proteins by electrophoresis resulted in a drastic reduction of nonspecific binding of labeled GTP to the filters. A serial dilution of colonies resulted in a single clone having GTP-binding activity. We checked about 60,000 colonies and detected two signals. This method is dependent on the ligand specificity of the expressed proteins and is different from the conventional methods based on DNA sequence similarity. This method can be used to isolate cDNAs encoding novel GTP-binding proteins.
Nagano
Y
Y
Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Japan.
Matsuno
R
R
Sasaki
Y
Y
D010582:Bacteriophage lambda / Q000235:genetics; D003001:Cloning, Molecular; D003101:Collodion; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000235:genetics*; D017343:Genes, Plant / Q000235:genetics; D006160:Guanosine Triphosphate / Q000378:metabolism; D015151:Immunoblotting / Q000379:methods; D008024:Ligands; D010940:Plant Proteins / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics
D004279:DNA, Viral; D008024:Ligands; D010940:Plant Proteins; D014764:Viral Proteins; D006160:Guanosine Triphosphate; D003101:Collodion; D004247:DNA; D019204:GTP-Binding Proteins