Expression and Purification of Wild and Mutant types of hCYP2S1 in E.coli to Study its Role in Drug Metabolism

CYP2S1, a drug metabolizing enzyme belonging to the superfamily of cyptochrome P450s. CYP2S1 is highly expressed in epithelial tissues and that are extra hepatic in origin. CYP2S1 expression is elevated in proliferative diseases such as breast, ovarian, and colorectal cancer and psoriasis, implying that it plays a role in cell proliferation. Recently it has been shown to be induced under Hypoxic conditions and the ability to metabolize an anti-cancer pro-drug AQ4N into an active drug AQ4. We will examine the single nucleotide polymorphisms (SNPs) to generate mutations for comparison to the wild type. We hypothesize there will be a significant difference between the wild and mutant variants of CYP2S1 in metabolic rates.  The purpose of this study is to perform Site Directed Mutagenesis with Polymerase Chain Reaction (PCR) on CYP2S1s’ single nucleotide polymorphisms (SNPs) including L189F, R166H, L230R, R380C, P446L and S61N. The SNPs were placed into an E.coli vector to be expressed and picked from XL-1 Blue competent cells to BL21DE-3 competent cells to be purified. The purification of CYP2S1 was done through High Performance Liquid Chromatography (HPLC) with a nickel column and Sodium Dodecyl Sulfate Polyacrylamide gel electrophoresis (SDS-PAGE) and Western blots were used for verification of the protein. Our lab was able to generate all SNPs and have one SNP, S61N, sequenced verified as a mutation. For further studies, the SNPs will be sequence verified for purification to continue the comparison of metabolic rates for known substrates and the innovation for others.  Research supported by R25 GM048998-13.