원문정보
초록
영어
Digestive carboxypeptidases are exopeptidases synthesized by the pancreas in the form of inactive zymogens called procarboxypeptidase. They are activated through limited proteolysis in the duodenum upon scission of about 100-residue N-terminal prosegment. The cDNA for the human pancreatic procarboxypeptidase B(hproCPB) was amplified by PCR and subcloned in the
vector pPIC9, which uses the methanol oxidase promoter and drives the high-level expression of heterologous proteins in P. pastoris. The recombinant plasmid was named as pPIC9-hproCPB(9.2kb). By digestion with SacI, the linearized pPIC9-hproCPB was transformed into P. pastoris strains GS115 and integrated into its genome. In the fermentor batch culture of Pichia transformed
cell on BMY medium, the activity of carboxypeptidase B after removal of pro region by trypsin treatment reached about 1.3 unit/ml. The fed-batch cultivation employing BMY medium and additional feeding of methanol gave about 11.7 unit/ml of hproCPB at 48 h. This result is corresponded to 10-fold higher level of enzyme activity than that of batch culture.