New strategies for the expression of human P‐glycoprotein (MDR1) in the yeast Pichia pastoris

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P‐glycoprotein (P‐gp) overexpression is often correlated with multidrug‐resistance (MDR) of cancer. The search for P‐gp inhibitors as co‐therapeutics to combat MDR has had little success after decades of studies. We previously used high‐throughput in silico ligand docking studies and identified drug‐like compounds that reversed MDR in human cancer cells in culture. Effects on P‐gp ATPase hydrolysis activity was used as an initial biochemical screen for inhibitor effectiveness followed by assays of MDR‐reversal of a prostate cancer cell line ((Brewer et al. in Mol Pharmacol. 2014; Follit et al in Pharmacol Res Perspect. 2015). More recently, computationally identified compounds were shown to reverse MDR in cancer cells, but did not appear to inhibit ATP hydrolysis by isolated P‐gp. One reason may be that mouse P‐gp rather than human P‐gp was previously used in our biochemical tests. Expression of human P‐gp in Pichia pastoris in a biologically active form was previously reported, but unfortunately, we experienced very modest expression levels of the human P‐gp expression construct for P. pastoris available in our lab. Codon optimization of mouse P‐gp was previously shown by others to result in increased protein yield, therefore a gene for human P‐gp was designed with optimized codon usage for translation in P. pastoris. A strong Kozak sequence was incorporated at the 5′ end of the coding frame. Unique internal restriction sites were incorporated that allow subcloning of individual sections of the gene, if necessary for troubleshooting and future mutagenesis. The DNA was synthesized using standard Gibson assembly methods and cloned into a pUC57 cloning vector. When transformed into E. coli, DNA rearrangement was observed. To identify whether a specific section of the insert was the cause of counter‐selection in E. coli, the insert DNA was digested into 5 component pieces and each one was subcloned. One of the sections was found to be more susceptible to rearrangements. We report here our attempts at constructing a stable, codon‐optimized, human P‐gp gene for high yield protein production in Pichia pastoris.Support or Funding InformationThis work is supported by NIH NIGMS [R15GM094771‐02] to JGW/PDV, SMU University Research Council, SMU Engaged Learning program, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas.