Construction of the plasmid PMEX8-HAK1 and random site-directed mutagenesis of human cytosolic adenylate kinase.
The pMEX8-hAK1 vector was devised from the pAK plasmid (Kim J. H. et al., 1989, Protein Engineering 5, 379-386), which could directly express human adenylate kinase proteins without recombination and its single strand DNA could be withdrawn with helper phage for random site-directed mutagenesis. The conserved key residues at Lys21, Lys27, and Thr39 were engineered to obtain mutants for kinetic analysis. Three mutants were obtained as K21P, K27R, and T39S, their specific activities were strikingly reduced compared to those of wild type adenylate kinase. This pMEX8-hAK1 will be a powerful tool for site-directed mutagenesis to detect the substrate-enzyme interaction for human adenylate kinase including various other enzymes.
Ayabe
T
T
Department of Hygiene, Miyazaki Medical College, Japan.
Takenaka
H
H
Takenaka
O
O
Takenaka
A
A
Nagahama
H
H
Maruyama
H
H
Yamamoto
A
A
Nagata
M
M
Koga
Y
Y
Sumida
M
M
Hamada
M
M
D000263:Adenylate Kinase / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D003600:Cytosol / Q000201:enzymology; D004252:DNA Mutational Analysis; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics*; D013379:Substrate Specificity
D000263:Adenylate Kinase