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Updated: Aug 18, 2026

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris
Published on: February 25, 2010
Multiple gene copy number enhances insulin precursor secretion in the yeast Pichia pastoris
Manuel Mansur1, Cecilia Cabello, Lester Hernández
1Center for Genetic Engineering and Biotechnology (CIGB), P.O. Box 6162, Havana 11600, Cuba. manuel.mansur@cigb.edu.cu
Abstract:
We have found a direct relationship between protein production in Pichia pastoris and the number of introduced synthetic genes of miniproinsulin (MPI), fused to the Saccharomyces cerevisiae pre-pro alpha factor used as secretion signal, and inserted between the alcohol oxidase 1 (AOX1) promoter and terminator sequences. Two consecutive approaches were followed to increase the number of integrated cassettes: the head-to-tail expression cassette multimerization procedure and re-transformation with a dominant selection marker. This increased expression from 19 to 250 mg l(-1) when about 11 copies have been integrated. Further, the correct position of one of the disulphide bridges of the purified molecule was verified by digestion with Glu-C endoprotease, followed by mass spectrometry of the isolated fragments.
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