Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Secretory Overexpression and Characterization of Human Procarboxypeptidase B from Saccharomyces cerevisiae

Saccharomyces cerevisiae에서 Human Procarboxypeptidase B의 과발현 분비생산과 그 특성

  • Kim, Mi-Jung (Department of Biotechnology and Bioengineering, Dong-Eui University) ;
  • Kim, Mi-Jin (Department of Biomaterial Control, Dong-Eui University) ;
  • Lee, Jae-Hyung (Department of Biomaterial Control, Dong-Eui University) ;
  • Kim, Yeon-Hee (Department of Biotechnology and Bioengineering, Dong-Eui University) ;
  • Seo, Jin-So (Department of Agricultural Biotechnology, Seoul National University) ;
  • Nam, Soo-Wan (Department of Biotechnology and Bioengineering, Dong-Eui University)
  • 김미정 (동의대학교 생명공학과) ;
  • 김미진 (동의대학교 바이오물질제어학과) ;
  • 이재형 (동의대학교 바이오물질제어학과) ;
  • 김연희 (동의대학교 생명공학과) ;
  • 서진호 (서울대학교 농생명공학부) ;
  • 남수완 (동의대학교 생명공학과)
  • Published : 2008.03.28
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Abstract

The gene encoding human pancreatic pro-carboxypeptidase B (CPB) was cloned and fused to Saccharomyces cerevisiae mating factor alpha-1 secretion signal $(MF{\alpha}1)$, in which the transcription of $MF{\alpha}1$-pro-CPB was under the control of GAL10 promoter. The constructed plasmid $pY{\alpha}$-hproCPB(7.72 kb) was transformed into S. cerevisiae 2805. The recombinant human pro-CPB (hproCPB) was successfully expressed in S. cerevisiae after induction of galactose, and could be secreted into the culture medium. By analyses of SDS-PAGE and western blotting, the molecular weight of the purified hproCPB was estimated to be a 45.9kDa. The activity of extracellular hCPB after removal of pro-region by trypsin treatment reached about 10.16 unit/ml at batch culture of S. cerevisiae $2805/pY{\alpha}$-hproCPB for 60 h. Also, the Km value of partially purified recombinant hCPB is about 0.43 mM.

인간 췌장 유래 pro-carboxypeptidase B(CPB) 유전자를 클로닝한 후 GAL10 promoter 하류에 Saccharomyces cerevisiae mating factor alpha-1 secretion signal $(MF{\alpha}-1)$과 연결하여 $MF{\alpha}1$-pro-CPB를 구축하였다. 구축된 plasmid $pY{\alpha}$-hproCPB(7.72 kb)를 S. cerevisiae 2805에 형질 전환시켰다. 재조합 인간 proCPB(hproCPB)는 galactose 유도로 S. cerevisiae에서 성공적으로 발현되었고, 배양상등액으로 분비되었다. SDS-PAGE와 western blot 분석에 의해, 정제된 hproCPB 단백질이 약 45.9kDa임을 확인하였다 S. cerevisiae $2805/pY{\alpha}$-hproCPB의 발효조 회분배양 시 trypsin 처리에 의해 pro영역을 제거한 후 hCPB의 세포외 활성은 60시간째에 10.16 unit/mL이었다. 또한 재조합 hCPB의 Km 값은 약 0.43 mM 이었다.

Keywords

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