Insights into the prevalence and underlying causes of clonal variation through transcriptomic analysis in Pichia pastoris
File(s)
Published version
Author(s)
Aw, R
Barton, GR
Leak, DJ
Type
Journal Article
Abstract
Clonal variation, wherein a range of specific productivities of secreted proteins are observed from supposedly identical transformants, is an accepted aspect of working with Pichia pastoris. It means that a significant number of transformants need to be tested to obtain a representative sample, and in commercial protein production, companies regularly screen thousands of transformants to select for the highest secretor. Here, we have undertaken a detailed investigation of this phenomenon by characterising clones transformed with the human serum albumin gene. The titers of nine clones, each containing a single copy of the human serum albumin gene (identified by qPCR), were measured and the clones grouped into three categories, namely, high-, mid- and low-level secretors. Transcriptomic analysis, using microarrays, showed that no regulatory patterns consistently correlated with titer, suggesting that the causes of clonal variation are varied. However, a number of physiological changes appeared to underlie the differences in titer, suggesting there is more than one biochemical signature for a high-secreting strain. An anomalous low-secreting strain displaying high transcript levels that appeared to be nutritionally starved further emphasises the complicated nature of clonal variation.
Date Issued
2017-05-22
Date Acceptance
2017-04-29
Citation
Applied Microbiology and Biotechnology, 2017, 101 (12), pp.5045-5058
ISSN
0175-7598
Publisher
Springer Verlag
Start Page
5045
End Page
5058
Journal / Book Title
Applied Microbiology and Biotechnology
Volume
101
Issue
12
Copyright Statement
©The Author(s) 2017. This article is an open access publication
License URL
Subjects
Science & Technology
Life Sciences & Biomedicine
Biotechnology & Applied Microbiology
Pichia pastoris/Komagataella phaffii
Transcriptomic analysis/microarray
Clonal variation
Unfolded protein response
ER-associated degradation
Protein expression
UNFOLDED PROTEIN RESPONSE
CHO-CELLS
PATHWAY IDENTIFICATION
COPY NUMBER
YEAST
EXPRESSION
GENES
INTEGRATION
ANNOTATION
AUTOPHAGY
Publication Status
Published