Purification and biochemical characterization of non-myristoylated recombinant pp60c-src kinase.
To obtain sufficient material for the biochemical and biophysical study of pp60c-src, we have utilized a recombinant pp60c-src baculovirus lacking the myristoylation site at codon 2. On infection of Sf9 cells, this virus produced large amounts of soluble non-myristoylated pp60c-src. The use of non-myristoylated pp60c-src (1) increases production of pp60c-src compared with the wild-type protein, (2) facilitates purification, (3) yields a stable product and (4) allows biochemical studies in the absence of detergents. Up to 20 mg of pp60c-src of greater than 95% purity has been purified from 6 litres of Sf9 cells grown in a bioreactor. One major and multiple minor forms of pp60c-src were separated by Mono Q f.p.l.c. Isoelectric focusing of purified pp60c-src species revealed heterogeneity, some of which could be attributed to differences in the tyrosine phosphorylation state of the enzyme. Kinetic analysis of non-myristoylated pp60c-src kinase in the presence of Mg2+ gave Km values for angiotensin II and ATP of 2 mM and 30 microM respectively and a Vmax. of 620 nmol/min per mg. The kinetic constants and metal ion preferences of a number of copolymers and peptide substrates have been compared. Polylysine and poly(GLAT), which was not phosphorylated by the pp60c-src kinase, dramatically activated autophosphorylation of Tyr-416, suggesting a conformation modulation of pp60c-src by charged polymers. This finding implies that Tyr-527 dephosphorylation is not sufficient for full activation of pp60c-src in vitro.
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D002460:Cell Line; D003001:Cloning, Molecular; D015151:Immunoblotting; D007700:Kinetics; D008969:Molecular Sequence Data; D009036:Moths; D016296:Mutagenesis; D009227:Myristic Acids / Q000378:metabolism; D010766:Phosphorylation; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D009227:Myristic Acids; D011994:Recombinant Proteins; D016392:Proto-Oncogene Proteins pp60(c-src)