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

Paper ID Volume ID Publish Year Pages File Format Full-Text
18382 42720 2007 5 PDF Available
Title
Expression, purification and characterization of porcine pancreatic Carboxypeptidase B from Pichia pastoris for the conversion of recombinant human insulin
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.

Keywords
Carboxypeptidase B; Protein secretion; Pichia pastoris
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Expression, purification and characterization of porcine pancreatic Carboxypeptidase B from Pichia pastoris for the conversion of recombinant human insulin
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 40, Issue 3, 5 February 2007, Pages 476–480
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us