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Real‐time PCR‐based determination of gene copy numbers in Pichia pastoris
Author(s) -
Abad Sandra,
Kitz Kerstin,
Hörmann Astrid,
Schreiner Ulrike,
Hartner Franz S.,
Glieder Anton
Publication year - 2010
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.200900233
Subject(s) - pichia pastoris , biology , gene , yeast , genome , transformation (genetics) , computational biology , heterologous , pichia , genetics , gene expression , codon usage bias , gene dosage , copy number variation , recombinant dna
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.