Characterization of Recombinant Human Man_9-Mannosidase Expressed in Escherichia coli

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概要

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• Man_9-mannosidase is an α1, 2-specific exo-enzyme involved in the N-linked oligosaccharide processing pathway. In this study, the expression of the truncated gene encoding for the human Man_9-mannosidase(amino acids 140 to 625)in Escherichia coli facilitated further characterization of the enzyme. PCR primers were designed to isolate a truncated human kidney Man_9-mannosidase gene. The gene was fused to a T7 protein tag and six histidine residues at the N and C-terminal ends, respectively. The truncated Man_9-mannosidase enzyme was purified by affinity chromatography and ion exchange chromatography. Activity of the purified enzyme was examined by HPLC and pyridylaminated(PA) oligosaccharides as substrates. Results showed that Man_9 GlcNAc_2 is rapidly converted to Man_9GlcNAc_2. Substrate specificity was analyzed using different isomers of Man_6GlcNAc_2, Man_7GlcNAc_2 and Man_8GlcNAc_2. The truncated Man_9-mannosidase exhibited very little activity towards the α1, 2-linked terminal mannose residue on the α3 branch of the Man_9GlcNAc_2, a characteristic of the pig and calf liver Man_9-mannosidases. Based on the HPLC analysis of the reaction products using size-fractionation and reversed-phase columns, the pathway for Man_9GlcNAc_2 trimming was deduced. The recombinant enzyme had optimum activity at 37-40℃ and pH 6.5-7.0. Enzyme activity was inhibited by 100 μM deoxymannojirimycin(dMNJ). Expression of the human kidney Man_9-mannosidase gene in E.coli facilitated the detailed characterization of the enzyme which until now had not been expressed in amounts necessary for biochemical and physical analyses.

• 社団法人日本生物工学会の論文
• 1998-09-25

著者

• Fujiyama Kazuhito

  International Center for Biotechnology, Osaka University

• YOSHIDA Toshiomi

  International Center for Biotechnology, Osaka University

• SEKI Tatsuji

  International Center for Biotechnology, Osaka University

• Kazuhito Fujiyama

  International Center For Biotechnology Osaka University

• Fujiyama Kazuhito

  大阪大学生物工学国際交流センター

• Fujiyama Kazuhito

  International Center For Biotechnology Osaka Univ.

• Fujiyama Kazuhito

  Department Of Fermentation Technology Osaka University

• Fujiyama Kazuhito

  International Center For Biotechnology Osaka University

• Seki Tatsuji

  International Center For Biotechnology Osaka University

• Seki Tatsuji

  大阪大学生物工学国際交流センター

• Moran Daniel

  The International Center For Biotechnology Osaka University:(present Address)kobe Technical Center P

• Moran Daniel

  大阪大学生物工学国際交流センター

• Moran Daniel

  The International Center For Biotechnology Osaka University

• Yoshida Toshiomi

  The International Center For Biotechnology Osaka University

• Yoshida Toshiomi

  International Center For Biotechnology Osaka University

• 吉田 豊和

  岐阜大・工・生命工

• SAKURADANI SHUJI

  The International Center for Biotechnology, Osaka University

• MORAN DANIEL

  International Center of Biotechnology, Osaka University

• SAKURADANI SHUJI

  International Center of Biotechnology, Osaka University

• JUN KIM

  International Center of Biotechnology, Osaka University

• Sakuradani Shuji

  The International Center For Biotechnology Osaka University

• Jun Kim

  International Center Of Biotechnology Osaka University:(present Address)dae-sang

• Yoshida Toshiomi

  Internatioal Center For Biotechnology Osaka University

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