Efficient isolation of genes by using antibody probes.
A sensitive and general technique has been devised for the dual purposes of cloning genes by using antibodies as probes and isolating unknown proteins encoded by cloned DNA. The method uses an expression vector, lambda gt11 (lac5 nin5 cI857 S100), that permits insertion of foreign DNA into the beta-galactosidase structural gene lacZ and promotes synthesis of hybrid proteins. Efficient screening of antigen-producing clones in lambda gt11 recombinant cDNA libraries is achieved through lysogeny of the phage library in hflA (high-frequency lysogeny) mutant cells of Escherichia coli; lysogens produce detectable quantities of antigen on induction, even when plated at high cell densities. The vector is also designed to facilitate the isolation of proteins specified by previously cloned gene sequences. Hybrid proteins encoded by recombinant phage accumulate in strains defective in protein degradation (lon mutants) in amounts amenable to large-scale purification. Antibodies produced against the portion of the hybrid encoded by foreign DNA could in turn be used to isolate the native polypeptide from eukaryotic cells.
D000818:Animals; D000906:Antibodies; D010582:Bacteriophage lambda / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004274:DNA, Recombinant / Q000302:isolation & purification; D004279:DNA, Viral / Q000302:isolation & purification; D005814:Genes, Viral; D008242:Lysogeny; D051379:Mice; D008810:Mice, Inbred C57BL; D011817:Rabbits; D014162:Transfection
D000906:Antibodies; D004274:DNA, Recombinant; D004279:DNA, Viral