A color-based stable multi-copy integrant selection system for Pichia pastoris using the attenuated ADE1 and ADE2 genes as auxotrophic markers

• Published in: Bioengineered 2012-01, Vol.3 (1), p.32-37
• Main Authors: Du, Min, Battles, Michael B., Nett, Juergen H.
• Format: Article
• Language: English
• Subjects: ADE1; ADE2; auxotrophic marker; Binding; Biology; Bioscience; Calcium; Cancer; Cell; Cycle; Fungal Proteins - genetics; Landes; multi-copy integration; Organogenesis; Pichia - genetics; Plasmids - genetics; Promoter Regions, Genetic - genetics; Proteins; stable integration
• ISSN: 2165-5979; 2165-5987; 1949-1026
• DOI: 10.4161/bbug.3.1.17936

The methylotropic yeast Pichia pastoris has been used for more than two decades to successfully produce a large number of recombinant proteins. Currently, a wide variety of auxotrophic and drug based selection markers are employed to screen for clones expressing the protein of interest. For most proteins an increased copy number of the integrated plasmid results in higher levels of expression, but these multi-copy integrants can be unstable due to the propensity of P. pastoris for homologous recombination. Here we describe a multi-copy selection system based on ade1 and ade2 auxotrophic parent strains and the respective attenuated markers with truncated promoter regions. We show that for all four proteins we tested, the use of the attenuated markers leads to increased protein expression when compared with selection based on the full strength markers. The fact that the adenine auxotrophic strains grow more slowly than the complemented counterparts essentially ensures the stability of multi-copy integration. At the same time, the accumulation of a red dye in the auxotrophic strains also provides an easy, color-based selection for transformants with multiple copies.