Saccharomyces cerevisiae에서 Human Procarboxypeptidase B의과발현 분비생산과 그 특성
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The gene encoding human pancreatic pro-carboxypeptidase B (CPB) was cloned and fused to Saccharomyces cerevisiae mating factor alpha-1 secretion signal (MFα1), in which the transcription of MFα1-pro-CPB was under the control of GAL10 promoter. The constructed plasmid pYα-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 45.9 kDa. 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/pYα-hproCPB for 60 h. Also, the Km value of partially purified recombinant hCPB is about 0.43 mM.
Abstract
The gene encoding human pancreatic pro-carboxypeptidase B (CPB) was cloned and fused to Saccharomyces cerevisiae mating factor alpha-1 secretion signal (MFα1), in which the transcription of MFα1-pro-CPB was under the control of GAL10 promoter. The constructed plasmid pYα-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 45.9 kDa. 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/pYα-hproCPB for 60 h. Also, the Km value of partially purified recombinant hCPB is about 0.43 mM.
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