Strategies for cloning and expression of a codon‐optimized human P‐glycoprotein (MDR1) in the yeast Pichia pastoris

Abstract

P‐glycoprotein (P‐gp) is an ABC transporter which actively transports xenobiotics out of cells. Overexpression of P‐gp is one of the main causes for the development of multidrug resistances (MDR) in cancer and other diseases. Because of its ability to bind and transport a large variety of chemically different compounds, overexpression of P‐gp in these MDR cancers leads to resistances against chemotherapeutic drugs that are distinctly different in structure, chemical composition, and functionality. Using high‐throughput in silico ligand docking studies, we have previously identified drug‐like compounds that inhibit P‐gp and cause reversal of MDR in a prostate cancer cell line (Brewer et al., Mol. Pharmacol. 86, 716–726, 2014; Follit et al., Pharmacol. Res. Perspect. 3, e00170, 2015). To analyze the mechanism of inhibition by these initially identified P‐gp inhibitors as well as newly discovered drug‐like compounds that reversed MDR in cancers, biochemical and biophysical analysis of inhibitor action is imperative. For this reason, a gene for human P‐gp (MDR1) was designed with optimized codon usage for translation in P. pastoris. A pPinkHC‐MDR1 plasmid was created for integration into Pichia pastoris PichiaPink™, a commercially available high yield expression system that includes specific strains with protease knock outs to limit protein degradation. Successful MDR1 DNA integration in the PichiaPink™ chromosome was confirmed, but significant protein expression has thus far not been observed. We have re‐cloned the codon optimized MDR1 DNA into another Pichia expression vector, pHIL. This vector allows integration of the gene of interest into the Pichia pastoris strain, GS115, which has been used successfully by us in the past to express a mouse cysteine‐less P‐glycoprotein. The resulting colonies displayed a Muts phenotype, which is indicative of the correct recombination into the Pichia genomic DNA. Initial Western blot analyses using both P‐gp specific and His‐tag specific antibodies showed that after 24 hours of growth in minimal methanol media the MDR1 GS115 clone did express reasonable amounts of human P‐gp. We report here our attempts at constructing a stable, codon optimized, human P‐gp gene for protein production in Pichia pastoris as well as attempts to express, purify and characterize the human P‐glycoprotein.Support or Funding InformationThis work is supported by NIH NIGMS [R15GM094771‐02] to John G. Wise, SMU University Research Council, SMU Hamilton Undergraduate Research Scholars and Undergraduate Research Assistantship Programs, the SMU Center for Drug Discovery, Design and Delivery, Ms. Lynn McBee and New England Biolabs, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.