Characterization of a panARS-based episomal vector in the methylotrophic yeast Pichia pastoris for recombinant protein production and synthetic biology applications.

BACKGROUND

production of recombinant proteins in the methylotrophic yeast Pichia pastoris largely depends on integrative vector. Although the stability of the integrated expression cassette was well rewarded for most applications, the availability of reliable episomal vector to host this would represent a useful tool to accelerate cloning and high-throughput screening, ameliorating also relatively high clonal variability reported in the transformants of integrative vector caused by the integration of off- targets in P. pastoris genome. Hamster Recombinant Proteins

More recently, heterologous and endogenous autonomously replicating sequences (ARS) have been identified in P. pastoris by genome mining, opening up the possibility of expanding the toolbox is available for efficient episomal plasmid insert. The purpose of this technical report is to validate the 452-bp sequence ( "panARS") in the context of P. pastoris expression vector, and to compare their performance to the classical integrative plasmid. In addition, we aimed to test whether episomal vectors such as would be suitable to sustain in vivo recombination, using fragments for transformation, directly in P. pastoris cells.

RESULTS

A vector-based episomal panARS evaluated using a blue fluorescent protein (BFP) as a reporter gene. Normalized fluorescence of colonies carry-BFP panARS surpass the level of the signals obtained from the control integrated by several-fold, while the endogenous sequence, identified from P. pastoris genome, inefficient in terms of protein production.

 At the level of single cells, clones panARS-BFP Mouse Recombinant Proteins showed lower variability but variations interclonal intraclonal higher than their integrated counterparts, supporting the idea that the production of heterologous proteins can benefit from episomal plasmid. Finally, the efficiency of 2-fragment and a 3-fragment in vivo recombination was tested with various lengths of overlapping regions and the molar ratio between the fragments. 

After optimization, minimal background vector obtained for in vivo assembly, show this could be a quick and efficient method to generate attractive episomal plasmid.

CONCLUSION

An expression vector based on the order panARS proven to outperform peers in terms of productivity integrative and variability interclonal protein, facilitating the expression of recombinant proteins and filtering. 

Using optimized fragment length and ratio, it is possible to perform a reliable in vivo recombination of fragments in P. pastoris. Taken together, these results support the application of panARS episomal vectors for synthetic biology approaches.