title: The PARS sequence increase the efficiency of stable Pichia pastoris transformation.
abstract: The methylotrophic yeast Pichia pastoris is a popular host for recombinant expression of proteins. Plasmids containing the Pichia autonomously replicating sequence (PARS) transform P. pastoris with higher efficiency than linear DNA equipped with termini designed for homologous recombination. Moreover, PARS containing constructs provide higher protein yields. Unfortunately, these autonomous plasmids are inherently unstable and the preferred method of P. pastoris transformation is therefore stable integration in the genome by homologous recombination. In the present study we report that a novel combination of PARS and linearization of plasmids for P. pastoris transformation serves to significantly increase the transformation efficiency. Moreover, it is demonstrated that the constructs do not re-circularize but integrate stably into the P. pastoris genome.
authors.affiliation: Department of Molecular Biology and Genetics, Section for Crop Genetics and Biotechnology, Aarhus University, Forsogsvej 1, 4200 Slagelse, Denmark. Electronic address: ClausKrogh.Madsen@mbg.au.dk.
authors.forename: Claus Krogh
authors.initials: CK
authors.lastname: Madsen
authors.affiliation: Wageningen University, 6708PB Wageningen, The Netherlands.
authors.forename: Gilles
authors.initials: G
authors.lastname: Vismans
authors.affiliation: Department of Molecular Biology and Genetics, Section for Crop Genetics and Biotechnology, Aarhus University, Forsogsvej 1, 4200 Slagelse, Denmark.
authors.forename: Henrik
authors.initials: H
authors.lastname: Brinch-Pedersen
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D005822:Genetic Vectors; D016681:Genome, Fungal; D059765:Homologous Recombination; D010843:Pichia / Q000235:genetics*; D010957:Plasmids; D011994:Recombinant Proteins; D018741:Replication Origin; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014170:Transformation, Genetic
chemical_list: D011994:Recombinant Proteins
keywords: Homologous recombination; PARS; Pichia pastoris; Transformation efficiency