An E. coli expression system for the rapid purification and characterization of a v-abl tyrosine protein kinase.
A bacterial expression vector containing a segment of the v-abl gene from Abelson murine leukemia virus (A-MuLV) was constructed such that the gag region of v-abl was replaced by a sequence encoding the IgG-binding domain of the S. aureus protein A. pabl HP, a fusion protein encoded by this vector was rapidly purified to near homogeneity by affinity chromatography on IgG-Affigel and Mono Q FPLC. The Km of the pabl HP kinase for ATP varied with [Val5]-angiotensin II concentration and was 21.2 microM at saturating concentrations of [Val5]-angiotensin II. The Km for [Val5]-angiotensin II at saturating concentrations of ATP was 3.8 mM. The turnover number, at 20 degrees C, was 62 mumol min-1 mumol-1. Initial rate studies support a ternary complex kinetic mechanism for phosphoryl transferase. The substrate specificity of the pabl HP kinase was further characterized using synthetic peptides. This expression system, which enables the rapid purification of recombinant v-abl kinase is suitable for the comparative enzymological study of mutant v-abl enzymes generated by site-directed mutagenesis.
Lydon
N B
NB
Research Department, Pharmaceutical Division, CIBA-GEIGY Limited, Basel, Switzerland.
Adams
B
B
Poschet
J F
JF
Gutzwiller
A
A
Matter
A
A
D000011:Abelson murine leukemia virus / Q000201:enzymology / Q000235:genetics*; D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D007700:Kinetics; D009052:Leukemia Virus, Murine / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016374:Oncogene Proteins v-abl; D009857:Oncogenes; D010455:Peptides / Q000138:chemical synthesis; D010766:Phosphorylation; D010957:Plasmids; D011505:Protein-Tyrosine Kinases / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D015682:Retroviridae Proteins, Oncogenic / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
D016374:Oncogene Proteins v-abl; D010455:Peptides; D011993:Recombinant Fusion Proteins; D015682:Retroviridae Proteins, Oncogenic; D011505:Protein-Tyrosine Kinases