Multiple gene copy number enhances insulin precursor secretion in the yeast Pichia pastoris

Mansur, M; Cabello, C; Hernandez, L; Pais, J; Varas, L; Valdes, J; Terrero, Y; Hidalgo, A; Plana, L; Besada, V; Garcia, L; Lamazares, E; Castellanos, L; Martinez, E

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.

Más información

Título según WOS: ID WOS:000228507600010 Not found in local WOS DB
Título de la Revista: BIOTECHNOLOGY LETTERS
Volumen: 27
Número: 5
Editorial: Springer
Fecha de publicación: 2005
Página de inicio: 339
Página final: 345
DOI:

10.1007/s10529-005-1007-7

Notas: ISI