title: Posttransformational vector amplification in the yeast Pichia pastoris.
abstract: Generating a high yield of recombinant protein is a major goal when expressing a foreign gene in any expression system. In the methylotrophic yeast Pichia pastoris, a common means of achieving this end is to select for transformants containing multiple integrated copies of an expression vector by plating them on high levels of a selectable marker drug followed by screening for rare colonies with multiple copies. We describe a more convenient method to select for such clones. Using Zeocin-resistance-based vectors, we demonstrate that strains transformed with only one or a few vector copies can, long after transformation, be subjected to further selection at high levels of drug. This resulted in the frequent selection of clones containing increased copy numbers of the vector. This posttransformational vector amplification (PTVA) process resulted in strains containing multiple head-to-tail copies of the entire vector integrated at a single locus in the genome. Of our PTVA selected clones, 40% showed a three- to fivefold increase in vector copy number. So-called 'jackpot' clones with >10 copies of the expression vector represented 5-6% of selected clones and had a proportional increase in recombinant protein.
authors.affiliation: Keck Graduate Institute of Applied Life Sciences, Claremont, CA 91711, USA.
authors.forename: Anthony Jay
authors.initials: AJ
authors.lastname: Sunga
authors.forename: Ilya
authors.initials: I
authors.lastname: Tolstorukov
authors.forename: James M
authors.initials: JM
authors.lastname: Cregg
mesh_terms: D001761:Bleomycin / Q000494:pharmacology; D025141:Drug Resistance, Fungal / Q000235:genetics*; D018274:Electroporation; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D018628:Gene Dosage; D015966:Gene Expression Regulation, Fungal; D005818:Genetic Engineering / Q000379:methods*; D005819:Genetic Markers / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D010843:Pichia / Q000187:drug effects / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D012641:Selection, Genetic; D014170:Transformation, Genetic; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D005819:Genetic Markers; D001761:Bleomycin; C105427:Zeocin; D001618:beta-Lactamases