Expression, purification and characterization of porcine pancreatic Carboxypeptidase B from Pichia pastoris for the conversion of recombinant human insulin

Mansur, M.; Martinez, L.; Perez, M.; Alonso-Del-Rivero, M.; Marquez, I.; Proenza, Y.; Varas, L.; Aviles, F., X.

Enzyme and Microbial Technology 40(3, Sp. Iss. SI): 476-480

2007


ISSN/ISBN: 0141-0229
DOI: 10.1016/j.enzmictec.2006.07.024
Accession: 012972495

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Abstract
Pancreatic or tissue Carboxypeptidase B (CPB), a key enzyme involved in insulin conversion and highly specific for excising C-terminal Lys and Arg residues from peptides and proteins, was expressed at high level and purified from a recombinant Pichia pastoris strain. A cDNA containing the porcine pancreatic pro-Carboxypeptidase B (proCPB) fused to the Saccharomyces cerevisiae alpha factor secretion signal was cloned into the pPIC3K vector under control of P pastoris AOX1 promoter. After 72 It of growth on methanol, proCPB accumulated until 320 mg L-1, representing 70% of total proteins in culture supernatant. A single stepwise ion exchange purification process with Q-Sepharose at increasing concentrations of ammonium acetate allowed recovery of 65% proCPB in a single fraction. The dialyzed protein was activated with trypsin and its activity was tested with the synthetic substrate Hippuryl-L-Arg. The kinetic parameters K-M and V-max, as well as inhibition constant K-i against a specific inhibitor were calculated and found similar to those of the wild-type enzyme. The enzyme efficiently removed amino acids Lys and Arg from the spacer of an insulin precursor (B1-30-LysArg-A(1-21)) expressed in yeast and previously cleaved with trypsin. The enzyme was found stable for 4 It at pH 11.8, a useful property for performing both enzyme reactions with trypsin and CPB in a single step.