Utilização do promotor do gene PGK1 de Pichia pastoris para expressão heteróloga

AUTOR(ES)
DATA DE PUBLICAÇÃO

2008

RESUMO

The methylotrophic yeast Pichia pastoris has been successfully used for the expression of heterologous proteins and several different expression vectors have been developed for this system. The main expression vectors for P. pastoris are based on the alcohol oxidase 1 gene promoter, AOX1. Some studies have been carried out aiming at the use of alternative promoters once the AOX1 system shows some disadvantages. Our laboratory has isolated and characterized the P. pastoris promoter from the PGK1 gene (PPGK1), a strong and constitutive promoter, which represents a promising alternative for the construction of new vectors. The aim of this study was to determine the minimal promoter sequences from PPGK1 through controlled deletions using the -amylase gene from Bacillus subtilis as a reporter gene. Four deletions were obtained and the smallest (PPGK 3) with ~ 400 bp, still maintained promoter activity. We have demonstrated that this sequence was able to direct vector integration into the P. pastoris genome and 51% of the transformants showed more than one copy integrated. The use of this promoter allowed high level expression of -amylase (250 UmL-1). Also, vectors for intracellular expression were based on the PGK promoter were also constructed which however still need to be improved for use in P. pastoris.

ASSUNTO(S)

pichia pastoris promotor - amilase promoter heterologous protein expression expressão heteróloga de proteínas biologia molecular pichia pastoris -amylase

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