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

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 72h of growth on methanol, proCPB accumulated until 320mgL−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 KM and Vmax, as well as inhibition constant Ki 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-A1–21) expressed in yeast and previously cleaved with trypsin. The enzyme was found stable for 4h at pH 11.8, a useful property for performing both enzyme reactions with trypsin and CPB in a single step.