Molecular Cloning, Nucleotide Sequence and Expression in Escherichia coli of Hyperthermophilic Glutamate Dehydrogenase Gene from Thermococcus profundus
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概要
- 論文の詳細を見る
The glutamate dehydrogenase (GDH) gene of a hyperthermophilic archaeon, Thermococcus profundus DT5432,was clned in Escherichia coli and sequenced. Analysis of the nucleotide sequence revealed an open reading frame of 1257 bp encoding a polypeptide of 419 amino acids with a molecular weigth of 46,699. The structura gene is preceded by an archaeal promoter consensus sequence with a spacing of 50 bases and is followed by a pyrimidine rich sequence which may function as a transcriptional terminator. The GDH gene was expressed in E. coli under control of the lac promoter. The purified recombinant GDH exhibited pH optima and thermostability identical to those of the intrinsic enzyme; however, it showed slightly higher K_m values for NADP, NADP^+, 2-oxoglutarate and ammonia than the intrinsic enzyme. The NH_2-terminal methionine was not removed in E. coli. The derived amino acid sequence is 53% identical to that of GDH from a mesophile, Clostridium difficile. Amino acid sequence alignment between the thermococcal and the clostridial GDH_s revealed the presence of significantly more alanine residues and significantly fewer glycine residues in α-helical regions, and more proline residues in loop regions, in the former than in the latter. These amino acid differences are located exclusively in the NH_2-terminal half (residues 54-253) of the enzyme.
- 社団法人日本生物工学会の論文
- 1997-05-25
著者
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Kobayashi Tetsuo
Laboratory of Gene Regulation, Graduate School of Bioagricultural Sciences, Nagoya University
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Kudo Toshiaki
Environmental Molecular Biology Lab. :graduate School Of Integrated Science Yokohama City University
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Kudo Toshiaki
Laboratory Of Microbiology The Institute Of Physical And Chemical Research(riken)
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Kawano H
Extremobiosphere Research Center (xbr) Japan Agency For Marine-earth Science And Technology (jamstec
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Takio K
Enviromental Molecular Biology Lab. Riken
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Kudo T
Riken (inst. Physical And Chemical Res.) Wako Jpn
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Kudo T
Riken Saitama Jpn
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Kudo Toshiaki
International Graduate School Of Arts And Sci. Yokohama City Univ.
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Kudo Toshiaki
Yokohama City Univ. Kanagawa Jpn
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HIGUCHI SADAHARU
Laboratory of Biochemistry, College of Science, Rikkyo (St. Paul's) University
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KIMURA KINUKO
Laboratory of Biochemistry, College of Science, Rikkyo(St. Paul's) University
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HORIKOSHI KOKI
DEEP-STAR Group, Japan Marine Science and Technology Ceter
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Kobayashi Tetsuo
Dep. Of Biological Mechanisms And Functions Graduate School Of Bioagricultural Sciences Nagoya Univ.
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Kudo Toshiaki
Laboratory Of Microbiology The Institute Of Physical And Chemical Research
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Horikoshi K
Extremobiosphere Res. Center (xbr) Japan Agency For Marine-earth Sci. And Technol. (jamstec)
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Kimura K
Laboratory Of Biochemistry College Of Science Rikkyo(st. Paul's) University
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Kobayashi T
Laboratory Of Microbiology The Institute Of Physical And Chemical Research (riken)
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Kobayashi T
Department Of Biological Mechanisms And Functions Graduate School Of Bioagricultural Sciences Nagoya
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Kobayashi Tetsuo
Laboratory Of Gene Regulation Graduate School Of Bioagricultural Sciences Nagoya University
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Higuchi Sadaharu
Laboratory Of Biochemistry College Of Science Rikkyo (st. Paul's) University
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Horikoshi Koki
Deep-sea Microorganisms Research Group Japan Marine Science And Technology Center
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Kimura Kinuko
Laboratory of Biochemistry, College of Science, Rikkyo(St. Paul's) University
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Kobayashi Tetsuo
Laboratory of Agricultural Meteorology, Faculty of Agriculture, Kyushu University
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