Abstract
Cytochrome P450 1B1 (CYP1B1) is a universal cancer marker and is implicated in many other disorders. Mutations in CYP1B1 are also associated with childhood blindness due to primary congenital glaucoma (PCG). To understand the CYP1B1 mediated etiopathology of PCG and pathomechanism of various cancers, it is important to carry out its functional studies. Heterologous expression of CYP1B1 in prokaryotes is imperative because bacteria yield a higher amount of heterologous proteins in lesser time and so the expressed protein is ideal for functional studies. In such expression system there is no interference by other eukaryotic proteins. But the story is not that simple as expression of heterologous CYP1B1 poses many technical difficulties. Investigators have employed various modifications/deletions of CYP N-terminus to improve CYP1B1 expression. However, the drawback of these studies is that it changes the original protein and, as a result, invalidates functional studies. The present study examines the role of various conditions and reagents in successful and consistent expression of sufficient quantities of unmodified/native human CYP1B1 in E. coli. We aimed at expressing CYP1B1 in various strains of E. coli and in the course developed a protocol that results in high expression of unmodified protein sufficient for functional/biophysical studies. We examined CYP1B1 expression with respect to different expression vectors, bacterial strains, types of culture media, time, Isopropyl β-D-1-thiogalactopyranoside concentrations, temperatures, rotations per minute, conditioning reagents and the efficacy of a newly described technique called double colony selection. We report a protocol that is simple, easy and can be carried out in any laboratory without the requirement of a fermentor. Though employed for CYP1B1 expression, this protocol can ideally be used to express any eukaryotic membrane protein.
Highlights
Cytochrome P450, family 1, subfamily B, polypeptide 1 (CYP1B1) is a recently identified [1] dioxin inducible aryl hydrocarbon hydroxylase with the enzyme commission number EC.1.14.14.1
Though we report the expression of Cytochrome P450 1B1 (CYP1B1) in this study but the protocol can, in principle, be applied to heterologous expression of any eukaryotic membrane protein
CYP1B1 expression is difficult in this regard and, several studies have been carried out using N-terminal modified protein [22,23,25,26,27,28,29]
Summary
Cytochrome P450, family 1, subfamily B, polypeptide 1 (CYP1B1) is a recently identified [1] dioxin inducible aryl hydrocarbon hydroxylase with the enzyme commission number EC.1.14.14.1. It catalyses the following master reaction: R-H z O2 z NAD(P)H z H ?R-OH z H2O z NAD(P). Being a member of the xenobiotics metabolizing family, CYP1B1 catalyzes the bioconversion/activation of a large number of procarcinogens but the reaction has a unique stereoselectivity and estradiol - 4 - hydroxylation is the characteristic of its catalytic actions [2]. CYP1B1 is different from both CYP1A1 and CYP1A2 in many respects It has only 40% homology with both these genes [2]. The expression of unmodified CYP1B1 is crucial for understanding its catalytic activities, cellular functions, molecular biology and the etiopathomechanistic aspects of the diseases it is involved in
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