Cloning, sequencing, and expression in Escherichia coli of cDNA encoding porcine brain UMP-CMP kinase.
A cDNA encoding porcine brain UMP-CMP kinase has been isolated using two oligonucleotide probes synthesized on the basis of the partial amino acid sequences of the purified enzyme. The isolated cDNA consisted of 1,626 nucleotides including the coding region for a polypeptide of 196 amino acid residues with a calculated molecular weight of 22,279. The enzyme showed an overall sequence identity of about 40 and 50%, respectively, with adenylate kinases from mammalian muscle and Escherichia coli and UMP-CMP kinases from Saccharomyces cerevisiae and Dictyostelium discoideum. The two highly conserved residues, Thr-39 and Leu-66, in adenylate kinases, which are located close to the adenine ring of the bound AMP, are replaced by Ala and Ile, respectively, at the corresponding positions in UMP-CMP kinases. The entire structural gene was inserted 3'-downstream of the strong promoter in the expression plasmid pET-3b. E. coli BL21(DE3) cells carrying the resultant plasmid produced the active enzyme in a soluble state, most efficiently upon induction at 37 degrees C with 0.02 mM isopropyl-beta-D-thiogalactoside. The purified recombinant enzyme catalyzed specific phosphoryl transfer from ATP to UMP and CMP.
Okajima
T
T
Faculty of Agriculture, Kinki University, Nara.
Goto
S
S
Tanizawa
K
K
Tagaya
M
M
Fukui
T
T
Shimofuruya
H
H
Suzuki
J
J
D000263:Adenylate Kinase / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D018076:DNA, Complementary; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D009703:Nucleoside-Phosphate Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013552:Swine; D015003:Yeasts / Q000201:enzymology
D004269:DNA, Bacterial; D018076:DNA, Complementary; D011994:Recombinant Proteins; D000263:Adenylate Kinase; D009703:Nucleoside-Phosphate Kinase