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

Manuel Mansur, Liliam Martínez, Mariela Pérez, Maday Alonso-del-Rivero, Isvaloy Márquez, Yanay Proenza, Laura Varas, Francesc X. Avilés

Research output: Contribution to journalArticleResearchpeer-review

5 Citations (Scopus)

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 h 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 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 4 h at pH 11.8, a useful property for performing both enzyme reactions with trypsin and CPB in a single step. © 2006 Elsevier Inc. All rights reserved.
Original languageEnglish
Pages (from-to)476-480
JournalEnzyme and Microbial Technology
Volume40
Issue number3
DOIs
Publication statusPublished - 5 Feb 2007

Keywords

  • Carboxypeptidase B
  • Pichia pastoris
  • Protein secretion

Fingerprint Dive into the research topics of 'Expression, purification and characterization of porcine pancreatic Carboxypeptidase B from Pichia pastoris for the conversion of recombinant human insulin'. Together they form a unique fingerprint.

Cite this