Identification of cDNA clones that code for protein-tyrosine kinases by screening expression libraries with antibodies against phosphotyrosine.
A phage lambda gt11 human fibroblast cDNA expression library was screened with antibodies against phosphotyrosine to determine the feasibility of this approach as a method for the identification of clones that code for protein-tyrosine kinases. Several antibody positive clones were isolated. One clone also scored positive with degenerate oligonucleotides designed to identify sequences coding for protein-tyrosine kinases. This cDNA clone was partially sequenced and proved to be identical to part of lyn, a recently reported putative protein-tyrosine kinase. A portion of the cDNA was cloned into an inducible plasmid expression vector. Phosphorylation of many bacterial proteins on tyrosine was observed upon addition of inducing agent. The results demonstrate that this method of screening can identify cDNAs that encode active protein-tyrosine kinases and could prove useful for the identification of novel ones.
Lindberg
R A
RA
Molecular Biology and Virology Laboratory, Salk Institute, San Diego, California 92138.
Thompson
D P
DP
Hunter
T
T
D000595:Amino Acid Sequence; D000906:Antibodies; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism*; D005347:Fibroblasts / Q000201:enzymology; D015870:Gene Expression; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D009857:Oncogenes; D010750:Phosphoproteins / Q000032:analysis / Q000378:metabolism; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis* / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
D000906:Antibodies; D018076:DNA, Complementary; D010750:Phosphoproteins; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases