Multiple gene copy number enhances insulin precursor secretion in the yeast Pichia pastoris.
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.
Mansur
Manuel
M
Center for Genetic Engineering and Biotechnology (CIGB), P.O. Box 6162, Havana 11600, Cuba. manuel.mansur@cigb.edu.cu
Cabello
Cecilia
C
Hernández
Lester
L
País
José
J
Varas
Laura
L
Valdés
Jorge
J
Terrero
Yanet
Y
Hidalgo
Abdel
A
Plana
Liuba
L
Besada
Vladimir
V
García
Liudys
L
Lamazares
Emilio
E
Castellanos
Lila
L
Martínez
Eduardo
E
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
D004220:Disulfides; D010455:Peptides; D011384:Proinsulin; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase