OPTIMIZATION OF HETEROLOGOUS EXPRESSION OF HUMAN CYTOCHROME P4501B1

Abstract

CYP450 is a superfamily of heme-thiolate monooxygenase and P4501B1 (CYP1B1) is the largest known enzyme of this superfamily. CYP1B1 is expressed in extra hepatic tissues and is responsible for the oxidative metabolism of a broad range of xenobiotics and structurally diverse exogenous and endogenous substrates. CYP1B1 is involved in many disorders such as cancer and its mutations are related to irreversible vision loss like glaucoma (Primary congenital glaucoma). In this study, CYP1B1 and its two mutants M2, and M3 clones were transformed in two different bacterial strains (DH5 alpha and BL21) and expression was analyzed. For analyzing the expression, RNA was extracted and the CYP1B1 expression was checked by semiquantitative PCR. Expression was found to be low in DH5 alpha cells whereas optimization of expression failed in BL21 cells. A moderate level of expression was observed with both uninduced and β-D-1-thiogalactopyranoside induction (IPTG) induced samples, which shows that IPTG induction did not play any role in enhancing expression levels of CYP1B1 in these cells. For protein expression, total protein was extracted, and SDS-PAGE was used to analyze presence of CYP1B1 protein. A 60kDa protein band was visible in SDS-PAGE, showing expression of CYP1B1 protein. Wild type and mutants (M2 and M3) protein expression was found low but same level of expression was found in wild and mutant clones indicating that mutations present in clones do not affect protein stability in DH5 alpha strain of E. coli. Keywords: Heterologous expression, CYP1B1, DH5 alpha cells