Bacterial expression of an active tyrosine kinase from a protein A/truncated c-src fusion protein.
The carboxy-terminal half of the c-src protein fused to the protein A moiety was expressed in bacteria. The protein A/truncated c-src fusion protein, which does not have SH2 and SH3 domains, is found in the periplasmic space allowing for a simple one-step purification and demonstrated high efficiency in autophosphorylation and exogeneous substrate phosphorylation. The missense mutation at codon 294 (Ile-->Thr), which is located in the ATP-binding domain of the c-src, resulted in dramatic reduction of tyrosine kinase activity of the fusion protein. Using the fusion protein, we also revealed that staurosporin, a well-known kinase inhibitor, directly affects autophosphorylation of the C-terminal half of the c-src protein. This truncated c-src expression system provides a good source of enzyme for diverse experiments and is an ideal model for understanding the implication of structural alterations in the catalytic activity of the c-src kinase by site-directed mutagenesis experiments.
Saya
H
H
Department of Neuro-Oncology, University of Texas M.D. Anderson Cancer Center, Houston 77030.
Lee
P S
PS
Nishi
T
T
Izawa
I
I
Nakajima
M
M
Gallick
G E
GE
Levin
V A
VA
D000470:Alkaloids / Q000494:pharmacology; D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli; D015971:Gene Expression Regulation, Enzymologic; D015151:Immunoblotting; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010957:Plasmids; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013205:Staphylococcal Protein A / Q000235:genetics*; D019311:Staurosporine
D000470:Alkaloids; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D011505:Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); D019311:Staurosporine