High-level secretory expression of human procarboxypeptidase B by Fed-Batch cultivation of Pichia pastoris and its partial characterization.

The procpb gene encoding human procarboxypeptidase B (proCPB, GeneBank access code AJ224866) was cloned and its Pichia expression plasmid, pPIC9alpha/hproCPB (9.2 kb), was constructed, in which procpb was under the control of the AOX1 promoter and connected to the downstream of the mating factor alpha-1 (MFalpha1) signal sequence. The plasmid was linearized by digestion with SacI, and integrated into the genome of P. pastoris strain GS115. By culturing of Pichia transformant on methanol medium, the human proCPB was successfully expressed and secreted into the culture supernatant. Moreover, Western blot analysis of the extracellular proteins showed proCPB bands clearly at a molecular mass of 45 kDa, confirming the expression of proCPB with its right size. The CPB activity reached about 3.5 U/ml and 12.7 U/ml in the flask and fermentor batch cultures of Pichia transformant, respectively. No CPB enzyme activity was found in the intracellular fraction. When the fedbatch cultivation was performed with methanol and glycerol mixture as a feeding medium, the extracellular CPB activity was increased to 42.0 U/ml, which corresponds to a 3.3-fold higher level of CPB activity than that of batch culture. The Km and kcat values of recombinant human CPB enzyme for hippuryl-L-Arg as a substrate were estimated to be 0.16mM and 11.93 sec-1, respectively.