title: Multiple gene copy number enhances insulin precursor secretion in the yeast Pichia pastoris.
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.
authors.affiliation: Center for Genetic Engineering and Biotechnology (CIGB), P.O. Box 6162, Havana 11600, Cuba. manuel.mansur@cigb.edu.cu
authors.forename: Manuel
authors.initials: M
authors.lastname: Mansur
authors.forename: Cecilia
authors.initials: C
authors.lastname: Cabello
authors.forename: Lester
authors.initials: L
authors.lastname: Hernández
authors.forename: José
authors.initials: J
authors.lastname: País
authors.forename: Laura
authors.initials: L
authors.lastname: Varas
authors.forename: Jorge
authors.initials: J
authors.lastname: Valdés
authors.forename: Yanet
authors.initials: Y
authors.lastname: Terrero
authors.forename: Abdel
authors.initials: A
authors.lastname: Hidalgo
authors.forename: Liuba
authors.initials: L
authors.lastname: Plana
authors.forename: Vladimir
authors.initials: V
authors.lastname: Besada
authors.forename: Liudys
authors.initials: L
authors.lastname: García
authors.forename: Emilio
authors.initials: E
authors.lastname: Lamazares
authors.forename: Lila
authors.initials: L
authors.lastname: Castellanos
authors.forename: Eduardo
authors.initials: E
authors.lastname: Martínez
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics; D000595:Amino Acid Sequence; D001709:Biotechnology / Q000379:methods*; D003001:Cloning, Molecular; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D018628:Gene Dosage; D005821:Genetic Techniques; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry; D010843:Pichia / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011384:Proinsulin / Q000235:genetics* / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D013997:Time Factors
chemical_list: D004220:Disulfides; D010455:Peptides; D011384:Proinsulin; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase