Multiple gene copy number enhances insulin precursor secretion in the yeast Pichia pastoris.
Biotechnol Lett
; 27(5): 339-45, 2005 Mar.
Article
em En
| MEDLINE
| ID: mdl-15834796
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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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Pichia
/
Proinsulina
/
Biotecnologia
/
Técnicas Genéticas
Limite:
Humans
Idioma:
En
Revista:
Biotechnol Lett
Ano de publicação:
2005
Tipo de documento:
Article
País de afiliação:
Cuba
País de publicação:
Holanda