Synthetic genes for human muscle-type adenylate kinase in Escherichia coli |
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| Authors: | Kim, Hyo Joon Nishikawa, Satoshi Tanaka, Toshiki Uesugi, Seiichi Takenaka, Hitoshi Hamada, Minoru Kuby, Stephen A. |
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| Affiliation: | Faculty of Pharmaceutical Sciences, Osaka University 1-6 Yamadaoka, Suita, Osaka 565 1Department of Hygiene, Miyazaki Medical College Kiyotake, Miyazaki-gun, Miyazaki 889-16, Japan 2The Laboratory for the Study of Hereditary and Metabolic Disorders and the Department of Biological Chemistry and Medicine, University of Utah Salt Lake City, UT 84108, USA |
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| Abstract: | An artificial gene coding for the human muscle-type cytosolicadenylate kinase (hAK1) was chemically synthesized and directlyexpressed in Escherichia coli under the control of trp promoter.The DNA duplex of 596 bp was designed and constructed from 40oligonucleotide fragments of typically 30 nucleotides in length.Twelve unique restriction sites were fairly evenly spaced inthe synthetic gene to facilitate site-specific mutagenesis atany part of this recombinant protein. The genes for mutant hAK1(Tyr 95 Phe 95, Y95F hAK1; Arg 97 Ala 97, R97AhAK1) were constructed by cassette mutagenesis and utilizedrestriction sites incorporated in the hAK1 gene. The recombinanthAK1 was purified to homogeneity by a two-step chromatographicprocedure with a good yield, and showed the same adenylate kinaseactivity as that of authentic hAK1. preliminary kinetic studiesshow that the enzymatic activity (Vmax app,cor) |
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| Keywords: | adenylate kinase/ MgATP binding site/ point mutation/ protein engineering/ synthetic gene/ AMP binding site. |
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