Optimization of CYP27A1 recombinant protein expression

Abstract

Human mitochondrial cytochrome P450 27A1 is a monooxygenase enzyme that oxidizes bile acids and other sterol derivatives. The enzyme plays an important role in sterol metabolism and is a potential target for clinical therapies related to metabolic conditions and certain cancers. To support the development of such therapies, detailed structural and functional studies of the enzyme should be pursued. Producing large quantities of purified, recombinant enzyme would enable these studies. Recombinant production of human cytochrome P450 27A1 in E. coli is challenging due to the enzyme being membrane associated. This work explores the optimization of human cytochrome P450 27A1 expression in E. coli by systematically testing the effects of cell strain, expression temperature, concentrations of induction reagents, and expression times. Western blot analysis is used to investigate the effects of variable changes prior to purification. E. coli cell strain (switching to C41(DE3)) appears to have the largest positive effect on overall yield. Increasing δ-aminolevulinic acid concentration (induces heme synthesis) also leads to significantly increased yields. Decreasing expression time decreases the amount of higher order cytochrome P450 aggregates that are formed. The combination of these changes is a more robust expression protocol with three major advantages: decreased expression time, lower aggregate to monomer ratios, and increased overall yield.

Fig. 1.

CYP27A1 expression in DH5α (control strain) and BL21 derived strains C41(DE3) and C43(DE3). Western blot of cell lysates (N = 3 biological replicates) A) overlay of total protein stain signal (green) with conjugated donkey anti-mouse IgG secondary antibody signal (red), B) secondary antibody only. C) Replicates plotted with average ± SD. Quantification of CYP27A1 band signal. Significance determined by one-way ANOVA, *P ≤ 0.05, ns = P > 0.05. Control conditions: 1 mM IPTG, 1 mM δALA, 1 mg/mL arabinose, expression at 28 °C for 48 h.

Fig. 2.

Effect of additives on C41-CYP27A1 expression. A) Representative western blot of cell lysates (top), left: overlay of total protein stain signal (green) with conjugated donkey anti-mouse IgG secondary antibody signal (red), right: secondary antibody only. Western blot shown is CYP27A1 expression in the presence of varying [δALA]. N = 3 biological replicates plotted with average ± SD. B) Quantification of CYP27A1 band signal with 1 mM IPTG as [control]. C) Quantification of CYP27A1 band signal with 1 mg/mL arabinose as [control]. D) Quantification of CYP27A1 band signal with 1 mM δALA as [control]. Significance determined by one-way ANOVA, *P ≤ 0.05. *P ≤ 0.05, **P ≤ 0.01, ns = P > 0.05.

Fig. 3.

C41-CYP27A1 expression at 4 h at tested temperatures. Western blot of cell lysates (N = 3 biological replicates) A) overlay of total protein stain signal (green) with conjugated donkey anti-mouse IgG secondary antibody signal (red), B) secondary antibody only. C) Replicates plotted with average ± SD. Quantification of CYP27A1 band signal. Significance determined by one-way ANOVA, *P ≤ 0.05, **P ≤ 0.01, ns = P > 0.05. Control: C41-CYP27A1 expressed at 28 °C for 48hr.

Fig. 4.

CYP27A1 expression over time at 28 °C. A) Western blot of cell lysates (N = 3 biological replicates), left: overlay of total protein stain signal (green) with conjugated donkey anti-mouse IgG secondary antibody signal (red), right: secondary antibody only. B) Quantification of CYP27A1 monomeric band signal (arrow). C) Replicates plotted with average ± SD. Quantification of CYP27A1 aggregate band signal (bracket). Significance determined by one-way ANOVA, ***P ≤ 0.001 ****P ≤ 0.0001, ns = P > 0.05. D) Time correlation between aggregate to monomer band ratio.

Fig. 5.

CYP27A1 expression in DH5α compared to C41(DE3) with new expression parameters. Western blot of cell lysates (N = 3 biological replicates), A) overlay of total protein stain signal (green) with conjugated donkey anti-mouse IgG secondary antibody signal (red), B) secondary antibody only. C) Replicates plotted with average ± SD. Quantification of CYP27A1 band signal. Significance determined by Student’s t-test, ****P ≤ 0.0001.

Fig. 6.

Vitamin D₃ kinetics curve. Activity of 0.2 μM human CYP27A1 in the presence of increasing Vitamin D₃ concentrations with an equal concentration of bovine adrenodoxin reductase and a 10-fold greater concentration of human adrenodoxin. Data represents two experiments with three technical replicates each. The generated curve was fit to the Michaelis-Menten equation using GraphPad Prism version 10.4.1 (Boston, Massachusetts USA).

Fig. 7.

Carbon monoxide binding spectrum of CYP27A1 lysate from A) DH5α, protein expression 48 h, B) C41 (DE3), protein expression 4 h.