caa a22 4500 397494017 CHVBK 20180308164516.0 cr unu---uuuuu 161202e199505 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a124830 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a124830 Cloning, Sequencing, and Expression in Escherichia coli of cDNA Encoding Porcine Brain UMP-CMP Kinase [Elektronische Daten] [Toshihide Okajima, Sachio Goto, Katsuyuki Tanizawa, Mitsuo Tagaya, Toshio Fukui, Hiroshi Shimofuruya, Jiro Suzuki] 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°C with 0.02 mM isopropyl-β-d-thiogalactoside. The purified recombinant enzyme catalyzed specific phosphoryl transfer from ATP to UMP and CMP. © Oxford University Press Regular Papers nationallicence cDNA cloning nationallicence expression nationallicence nucleoside monophosphate kinase nationallicence nucleotide-binding loop nationallicence porcine brain nationallicence Okajima Toshihide Faculty of Agriculture, Kinki University Nara, Nara 631 aut Goto Sachio Faculty of Agriculture, Kinki University Nara, Nara 631 aut Tanizawa Katsuyuki School of Materials Science, Toyohashi University of Technology Toyohashi, Aichi 441 aut Tagaya Mitsuo Institute of Scientific and Industrial Research, Osaka University Ibaraki, Osaka 567 aut Fukui Toshio Institute of Scientific and Industrial Research, Osaka University Ibaraki, Osaka 567 aut Shimofuruya Hiroshi School of Materials Science, Toyohashi University of Technology Toyohashi, Aichi 441 aut Suzuki Jiro School of Materials Science, Toyohashi University of Technology Toyohashi, Aichi 441 aut The Journal of Biochemistry Oxford University Press 117/5(1995-05), 980-986 0021-924X 117:5<980 1995 117 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a124830 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a124830 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Okajima Toshihide Faculty of Agriculture, Kinki University Nara, Nara 631 aut NATIONALLICENCE 700 1- Goto Sachio Faculty of Agriculture, Kinki University Nara, Nara 631 aut NATIONALLICENCE 700 1- Tanizawa Katsuyuki School of Materials Science, Toyohashi University of Technology Toyohashi, Aichi 441 aut NATIONALLICENCE 700 1- Tagaya Mitsuo Institute of Scientific and Industrial Research, Osaka University Ibaraki, Osaka 567 aut NATIONALLICENCE 700 1- Fukui Toshio Institute of Scientific and Industrial Research, Osaka University Ibaraki, Osaka 567 aut NATIONALLICENCE 700 1- Shimofuruya Hiroshi School of Materials Science, Toyohashi University of Technology Toyohashi, Aichi 441 aut NATIONALLICENCE 700 1- Suzuki Jiro School of Materials Science, Toyohashi University of Technology Toyohashi, Aichi 441 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 117/5(1995-05), 980-986 0021-924X 117:5<980 1995 117 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford