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...

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Bibliographic Details
Published in:Biotechnology letters 2005-03, Vol.27 (5), p.339-345
Main Authors: MANSUR, Manuel, CABELLO, Cecilia, GARCIA, Liudys, LAMAZARES, Emilio, CASTELLANOS, Lila, MARTINEZ, Eduardo, HERNANDEZ, Lester, PAIS, José, VARAS, Laura, VALDES, Jorge, TERRERO, Yanet, HIDALGO, Abdel, PLANA, Liuba, BESADA, Vladimir
Format: Article
Language:English
Subjects:
Alcohol Oxidoreductases - genetics
Amino Acid Sequence
Biological and medical sciences
Biotechnology
Biotechnology - methods
Cloning, Molecular
Disulfides - chemistry
Electrophoresis, Polyacrylamide Gel
Fermentation
Fundamental and applied biological sciences. Psychology
Gene Dosage
Genetic Techniques
Genetic Vectors
Humans
Kinetics
Mass Spectrometry
Microbiology
Molecular Sequence Data
Peptide Mapping
Peptides - chemistry
Pichia - metabolism
Pichia pastoris
Plasmids - metabolism
Proinsulin - genetics
Proinsulin - secretion
Promoter Regions, Genetic
Saccharomyces cerevisiae
Saccharomyces cerevisiae - metabolism
Serine Endopeptidases - metabolism
Time Factors
Yeasts
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Summary: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.
ISSN:0141-5492
1573-6776
DOI:10.1007/s10529-005-1007-7