AIM AND OBJECTIVE: This study describes the design and evaluation of an expression vector for Pichia pastoris (pPICZáBHF), which is based on the commercial vector construct pPICZáB.MATERIAL AND METHODS: The performance of pPICZáBHF 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 pPICZáBHF plasmid for yeast cell transformation in comparison with the commercial vector pPICZáB, 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 pPICZáBHF 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 pPICZáBHF may be beneficial for the applications such as high-throughput screening of mutant gene libraries.
Gene Library Genetic Engineering