High level expression of chicken muscle adenylate kinase in Escherichia coli.
Chicken muscle adenylate kinase was produced in a large amount in Escherichia coli cells harboring an expression plasmid, pKK-cAKl-1. The plasmid was constructed by placing the cDNA sequence for chicken muscle adenylate kinase after the tac promoter. After induction by isopropyl-beta-D-thiogalactopyranoside, the enzyme protein amounted to about 10% of the bacterial proteins. The enzyme was readily purified in two steps by using phosphocellulose and Sephadex G-100 columns. The apparent molecular weight of the enzyme produced in E. coli was estimated to be 22,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in agreement with the value deduced from the cDNA sequence. Ten amino acids in the NH2-terminal region were determined, and were identical with the sequence deduced from the cDNA sequence except that the terminal methionine was absent. Michaelis constants for ATP, ADP, and AMP of the enzyme thus synthesized were essentially identical to those determined with the enzyme in crude extracts of chicken skeletal muscle.
Tanizawa
Y
Y
Third Department of Internal Medicine, School of Medicine, Yamaguchi University.
Kishi
F
F
Kaneko
T
T
Nakazawa
A
A
D000263:Adenylate Kinase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002645:Chickens; D004247:DNA / Q000032:analysis; D004790:Enzyme Induction; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D010770:Phosphotransferases / Q000235:genetics*; D010957:Plasmids
D007544:Isopropyl Thiogalactoside; D004247:DNA; D010770:Phosphotransferases; D000263:Adenylate Kinase