Real‐time PCR‐based determination of gene copy numbers in Pichia pastoris

Abstract

Pichia pastoris is a preferred host for heterologous protein production. Expression cassettes are usually integrated into the genome of this methylotrophic yeast. This manuscript describes a method for fast and reliable gene copy number determinations for P. pastoris expression strains. We believe that gene copy number determinations are important for all researchers working with P. pastoris and also many other research groups using similar gene integration techniques for the transformation of other yeasts. The described method uses real-time PCR to quantify the integrated expression cassettes. Similar methods were employed previously for other host systems such as animal and plant cells but no such method comparing different detection methods and describing details for yeast analysis by quantitative PCR is known to us, especially for methylotrophic yeasts such as P. pastoris. Neglecting gene copy numbers can easily lead to false interpretations of experimental results from codon optimization or promoter studies and co-expression of helper proteins as demonstrated in an application example, which is also described here.

Effect of Plasmid Design and Type of Integration Event on Recombinant Protein Expression in Pichia pastoris

“…10% (0.61% Ϯ 0.07%). The expected CN of 1 was slightly underestimated, but variation to this extent was also noted with quantitative PCR (qPCR) (48) CN determinations in P. pastoris (43,49,50). Also, relative conclusions on the CN differences between the strains were not influenced.…”

Species-Dependent Uptake of Glycylsarcosine but Not Oseltamivir in Pichia pastoris Expressing the Rat, Mouse, and Human Intestinal Peptide Transporter PEPT1

“…The GS115 yeast cells were then cultured on MM (1.34% YNB, 4 ϫ 10 Ϫ5 % biotin, 0.5% methanol, and 1.5% agarose) and MD (1.34% YNB, 4 ϫ 10 Ϫ5 % biotin, 2% dextrose, 1.5% agarose) plates for distinguishing the His ϩ Mut ϩ from His ϩ Mut s transformants. After isolation of genomic DNA from the His ϩ Mut ϩ GS115 transformants, real-time PCR was performed with species-specific primers (Table 3) to measure the gene copy number of Pept1 cDNA in yeast cells (Abad et al, 2010). The ARG4 gene of yeast was set as the internal control, and the plasmid DNA pPIC3.5K/Pept1 was set as the positive control.…”

High-level expression of Rhodotorula gracilis d-amino acid oxidase in Pichia pastoris

“…Two strains thought to feature single and multicopy gene integration events were chosen and are henceforth named RgDAO_sc and RgDAO_mc, respectively. Quantitative real-time PCR analysis (Abad et al 2010a) confirmed the presence of 1 (RgDAO_sc) and 12-13 (RgDAO_mc) copies of the expression cassette integrated in the P. pastoris genome.…”

“…After electroporation and regeneration for 2 h at 30°C using agitation at 60 rpm, aliquots (0.2 ml) were plated on solid selection medium containing 100 mg Zeocin/l. The number of copies of RgDAO expression cassettes integrated into the P. pastoris genome was determined by quantitative real-time PCR as described in Abad et al (2010a), employing the AOX1 promoter as target and the endogenous ARG4 gene as a reference.…”