Purification and biochemical characterization of non-myristoylated recombinant pp60c-src kinase.

Abstract

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.

• Authors: N B Lydon, B Gay, H Mett, B Murray, J Liebetanz, A Gutzwiller, H Piwnica-Worms, T M Roberts, E McGlynn
• Journal: The Biochemical journal (Biochem J) Vol. 287 ( Pt 3) Pg. 985-93 (Nov 01 1992) ISSN: 0264-6021 [Print] England
• PMID: 1280108 (Publication Type: Journal Article)

Chemical References

• Myristic Acids
• Recombinant Proteins
• Proto-Oncogene Proteins pp60(c-src)

Topics

• Amino Acid Sequence
• Animals
• Baculoviridae
• Cell Line
• Cloning, Molecular
• Immunoblotting
• Kinetics
• Molecular Sequence Data
• Moths
• Mutagenesis
• Myristic Acids (metabolism)
• Phosphorylation
• Proto-Oncogene Proteins pp60(c-src) (genetics, isolation & purification, metabolism)
• Recombinant Proteins (isolation & purification, metabolism)
• Substrate Specificity