Evaluation of a new episomal vector based on the GAP promoter for structural genomics in Pichia pastoris

초록

A new constitutive episomal expression vector, pGAPZ-E, was constructed and used for initial screening of eukaryotic target gene expression in Pichia pastoris. Two reporter genes such as beta-galactosidase gene and GFPuv gene were overexpressed in P. pastoris. The expression level of the episomal pGAPZ-E strain was higher than that of the integrated form when the beta-galactosidase gene was used as the reporter gene in P pastoris X33. The avoiding of both the integration procedure and an induction step simplified the overall screening process for eukaryotic target gene expression in P pastoris. Nine human protein targets from the Core 50, family of Northeast Structural Genomics Consortium (http://www.nesg.org), which were intractable when expressed in E. coli, were subjected to rapid screening for soluble expression in P pastoris. HR547, HR919, and HR1697 human proteins, which had previously been found to express poorly or to be insoluble in E. coli, expressed in soluble form in P pastoris. Therefore, the new episomal GAP promoter vector provides a convenient and alternative system for high-throughput screening of eukaryotic protein expression in P pastoris.

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