Title:Engineering of an Episomal Plasmid Suitable for High-Throughput Expression in Pichia pastoris
Volume: 20
Issue: 8
Author(s): Huan Chen, Cui Cao, Anna Kulinich, Li Liu*, Yong-Sam Jung*Josef Voglmeir*
Affiliation:
- Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University,China
- Department of Veterinary Medicine, Nanjing Agricultural University, Nanjing,China
- Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University,China
Keywords:
Expression vector, Pichia pastoris, Pichia-specific autonomous replication sequence, promoter, red fluorescent
protein, episomal expression.
Abstract: Aim and objective: This study describes the design and evaluation of an expression vector
for Pichia pastoris (pPICZαBHF), which is based on the commercial vector construct pPICZαB.
Material and Methods: The performance of pPICZαBHF was evaluated with red fluorescent protein
as a reporter. Additional His- and Flag-tags on the N-terminal ensured a simplified protein
purification procedure. Transformation efficiency, expression level and plasmid maintenance were
studied in order to test the functionality and usefulness of the constructed vector.
Results: We found that high transformation efficiencies were achieved using pPICZαBHF plasmid
for yeast cell transformation in comparison with the commercial vector pPICZαB, which has to be
integrated into the Pichia genome. However, expression levels of the recombinant protein were
generally lower compared to the commercial construct. Recombinant plasmids were shown to be
maintained in cells for approximately five days.
Conclusion: Although pPICZαBHF may not be suitable for the production of high levels of
recombinant protein, the simplicity of this P. pastoris expression system may still be of interest for
the expression of proteins involved in cofactor regeneration or the expression of reporter genes. In
addition, high transformation efficiency of pPICZαBHF may be beneficial for the applications such
as high-throughput screening of mutant gene libraries.
