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A novel methodology for enhanced and consistent heterologous expression of unmodified human cytochrome P450 1B1 (CYP1B1).

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 ?-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.

SUBMITTER: Faiq MA

PROVIDER: S-EPMC4199734 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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A novel methodology for enhanced and consistent heterologous expression of unmodified human cytochrome P450 1B1 (CYP1B1).

Faiq Muneeb A MA Ali Mashook M Dada Tanuj T Dada Rima R Saluja Daman D

PloS one 20141016 10

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 ...[more]

PMID: 25329831

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Project description:Previously, we showed that peripheral administration of 6β-hydroxytestosterone, a CYP1B1 (cytochrome P450 1B1)-generated metabolite of testosterone, promotes angiotensin II-induced hypertension in male mice. However, the site of action and the underlying mechanism by which 6β-hydroxytestosterone contributes to angiotensin II-induced hypertension is not known. Angiotensin II increases blood pressure by its central action, and CYP1B1 is expressed in the brain. This study was conducted to determine whether testosterone-CYP1B1 generated metabolite 6β-hydroxytestosterone locally in the brain promotes the effect of systemic angiotensin II to produce hypertension in male mice. Central CYP1B1 knockdown in wild-type (Cyp1b1+/+) mice by intracerebroventricular-adenovirus-GFP (green fluorescence protein)-CYP1B1-short hairpin (sh)RNA attenuated, whereas reconstitution of CYP1B1 by adenovirus-GFP-CYP1B1-DNA in the paraventricular nucleus but not in subfornical organ in Cyp1b1-/- mice restored angiotensin II-induced increase in systolic blood pressure measured by tail-cuff. Intracerebroventricular-testosterone in orchidectomized (Orchi)-Cyp1b1+/+ but not in Orchi-Cyp1b1-/-, and intracerebroventricular-6β-hydroxytestosterone in the Orchi-Cyp1b1-/- mice restored the angiotensin II-induced: (1) increase in mean arterial pressure measured by radiotelemetry, and autonomic imbalance; (2) reactive oxygen species production in the subfornical organ and paraventricular nucleus; (3) activation of microglia and astrocyte, and neuroinflammation in the paraventricular nucleus. The effect of intracerebroventricular-6β-hydroxytestosterone to restore the angiotensin II-induced increase in mean arterial pressure and autonomic imbalance in Orchi-Cyp1b1-/- mice was inhibited by intracerebroventricular-small interfering (si)RNA-androgen receptor (AR) and GPRC6A (G protein-coupled receptor C6A). These data suggest that testosterone-CYP1B1-generated metabolite 6β-hydroxytestosterone, most likely in the paraventricular nucleus via AR and GPRC6A, contributes to angiotensin II-induced hypertension and neuroinflammation in male mice.

Project description:Previously, we showed that peripheral administration of 2-ME (2-methoxyestradiol), a CYP1B1 (cytochrome P450 1B1)-catechol-O-methyltransferase (COMT) generated metabolite of E2 (17?-Estradiol), protects against angiotensin II-induced hypertension in female mice. The demonstration that central E2 inhibits angiotensin II-induced hypertension, together with the expression of CYP1B1 in the brain, led us to hypothesize that E2-CYP1B1 generated metabolite 2-ME in the brain mediates its protective action against angiotensin II-induced hypertension in female mice. To test this hypothesis, we examined the effect of intracerebroventricularly (ICV) administered E2 in ovariectomized (OVX)-wild-type (Cyp1b1+/+) and OVX-Cyp1b1-/- mice on the action of systemic angiotensin II. ICV-E2 attenuated the angiotensin II-induced increase in mean arterial blood pressure, impairment of baroreflex sensitivity, and sympathetic activity in OVX-Cyp1b1+/+ but not in ICV-injected short interfering (si)RNA-COMT or OVX-Cyp1b1-/- mice. ICV-2-ME attenuated the angiotensin II-induced increase in blood pressure in OVX-Cyp1b1-/- mice; this effect was inhibited by ICV-siRNA estrogen receptor-? (ER?) and G protein-coupled estrogen receptor 1 (GPER1). ICV-E2 in OVX-Cyp1b1+/+ but not in OVX-Cyp1b1-/- mice and 2-ME in the OVX-Cyp1b1-/- inhibited angiotensin II-induced increase in reactive oxygen species production in the subfornical organ and paraventricular nucleus, activation of microglia and astrocyte, and neuroinflammation in paraventricular nucleus. Furthermore, central CYP1B1 gene disruption in Cyp1b1+/+ mice by ICV-adenovirus-GFP (green fluorescence protein)-CYP1B1-short hairpin (sh)RNA elevated, while reconstitution by adenovirus-GFP-CYP1B1-DNA in the paraventricular nucleus but not in subfornical organ in Cyp1b1-/- mice attenuated the angiotensin II-induced increase in systolic blood pressure. These data suggest that E2-CYP1B1-COMT generated metabolite 2-ME, most likely in the paraventricular nucleus via estrogen receptor-? and GPER1, protects against angiotensin II-induced hypertension and neuroinflammation in female mice.

Dataset Information

A novel methodology for enhanced and consistent heterologous expression of unmodified human cytochrome P450 1B1 (CYP1B1).

Publications

A novel methodology for enhanced and consistent heterologous expression of unmodified human cytochrome P450 1B1 (CYP1B1).

Similar Datasets

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