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Engineering of an Episomal Plasmid Suitable for High-Throughput Expression in Pichia pastoris

Author(s):

Huan Chen, Cui Cao, Anna Kulinich, Li Liu, Yong-Sam Jung and Josef Voglmeir   Pages 1 - 8 ( 8 )

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 Method: The performance of pPICZαBHF was evaluated with red fluorescence protein as a reporter. Additional His- and Flag-tags on the N-terminal ensure 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 suitable for the production of high levels of recombinant protein, the simplicity of this P. pastoris expression system may be still 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 the high-throughput screening of mutant gene libraries.

Keywords:

expression vector, Pichia pastoris, Pichia-specific autonomous replication sequence, promoter, red fluorescent protein, episomal expression.

Affiliation:

Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Department of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Glycomics and Glycan Bioengineering Research Center (GGBRC), College of Food Science and Technology, Nanjing Agricultural University, Nanjing



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