title: An episomal expression vector for screening mutant gene libraries in Pichia pastoris.
abstract: Screening mutant gene libraries for isolating improved enzyme variants is a powerful technique that benefits from effective and reliable biological expression systems. Pichia pastoris is a very useful organism to express proteins that are inactive in other hosts such as Escherichia coli and Saccharomyces cerevisiae. However, most P. pastoris expression plasmids are designed to integrate into the host chromosome and hence are not as amenable to high-throughput screening projects. We have designed a P. pastoris expression vector, pBGP1, incorporating an autonomous replication sequence that allows the plasmid to exist as an episomal element. This vector contains the alpha-factor signal sequence to direct secretion of the mutant enzymes. Expression of the genes is driven by the constitutive GAP promoter, thus eliminating the need for timed or cell density-specific inductions. The pBGP1 plasmid was used to screen a xylanase gene library to isolate higher activity mutants.
authors.affiliation: USDA-ARS-WRRC, 800 Buchanan St., Albany, CA 94710, USA. clee@pw.usda.gov
authors.forename: Charles C
authors.initials: CC
authors.lastname: Lee
authors.forename: Tina G
authors.initials: TG
authors.lastname: Williams
authors.forename: Dominic W S
authors.initials: DW
authors.lastname: Wong
authors.forename: George H
authors.initials: GH
authors.lastname: Robertson
mesh_terms: D001761:Bleomycin / Q000494:pharmacology; D004261:DNA Replication / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D015723:Gene Library; D005822:Genetic Vectors / Q000187:drug effects / Q000235:genetics*; D009154:Mutation; D010843:Pichia / Q000187:drug effects / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014995:Xylosidases / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D001761:Bleomycin; C105427:Zeocin; D014995:Xylosidases