A Novel Methodology for Enhanced and Consistent Heterologous Expression of Unmodified Human Cytochrome P450 1B1 (CYP1B1)

<div><p>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 <i>E. coli</i>. We aimed at expressing CYP1B1 in various strains of <i>E. coli</i> 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.</p></div

Composition of the trace element solution and the various combinations of terrific broth used in the experimental setup.

<p>Composition of the trace element solution and the various combinations of terrific broth used in the experimental setup.</p

Panoramic view of the experiments carried out with the numerical values of the variables and the results obtained.

<p>The best fit in each variable is also shown in the rightmost column.</p><p>Panoramic view of the experiments carried out with the numerical values of the variables and the results obtained.</p

PAGE/Western blot pictures of CYP1B1 expression for various variables with absorption spectrum adjoining.

<p>Each unit shows absorption spectrum of the expressed protein at 350 to 600 nm. Each absorption spectrum shows a peak somewhere around 410–420 nm which is characteristic of the heme present in the CYP1B1 protein. The colours in each graph has a defined sequence which correlates with the SDS-PAGE picture shown just underneath each absorption curve. In every graph, the dark red colour corresponds to the second lane of the gel picture underneath (lane next to marker which is the first lane starting from the left). The dark green corresponds to the third lane, dark blue to the fourth, cyan colour to the fifth, sixth is pink and seventh is the fluorescent green. (<b>A</b>) CYP1B1 expression with three different vectors viz. pET28a+, pCWori(+) and pTRC-His. (<b>B</b>) CYP1B1 expression with different media viz. LB-broth, tryptone based TB, peptone based TB and TB made with 1∶1 mixture of tryptone and peptone. (<b>C</b>) CYP1B1 expression at different time points viz. 12 h, 20 h, 24 h and 30 h (<b>D</b>) CYP1B1 expression with six different strains of <i>E. coli</i> viz. DH5α, JM109, C100, DE3, Codon Plus and Pril. (<b>E</b>)Western blot showing the increase in protein expression with increasing IPTG concentration. Starting from left is marker folowed by expression without IPTG induction and then folowed by 0.2 mM, 0.4 mM, 0.6 mM and 0.8 mM IPTG concentrations. (<b>F</b>) Expression pattern of CYP1B1 at four different temperatures viz. 18°C, 25°C, 30°C and 37°C. (<b>G</b>) CYP1B1 expression without and with TES. (<b>H</b>) CYP1B1 expression pattern with different concentrations of thiamine viz. 0.5 mM, 1.0 mM and 1.5 mM. (<b>I</b>) Expression pattern of CYP1B1 at different δ-ALA concentrations viz. 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM and 1.0 mM. (<b>J</b>) CYP1B1 expression with and without double colony selection. (<b>K</b>) Expression pattern of CYP1B1 at four different rpm levels viz. 150, 170, 200, 250. (<b>L</b>) Overall experiment layout with all the variables and results obtained.</p

Schematic Diagram of CYP1B1 gene.

<p>The CYP1B1 gene has 2 introns and 3 exons. The open reading frame starts from Exon II and ends within Exon III. The gene codes for a 543 aminoacid protein with a membrane spanning domain at the N-terminal followed by a proline rich hinge region which in turn is followed by the cytosolic globular domain. The chromosomal location of the gene is 2p21–22.</p

Study recruitment profile.

<p>Study recruitment profile.</p

Multivariable logistic regression analysis for risk factors for NDDs^#.

<p>Multivariable logistic regression analysis for risk factors for NDDs<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.1002615#t005fn001" target="_blank">^#</a>.</p

Prevalence estimates of NDDs for the five study districts according to age categories^*.

<p>Prevalence estimates of NDDs for the five study districts according to age categories^{<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.1002615#t004fn001" target="_blank">*</a>}.</p

Background characteristics of study participants.

<p>Background characteristics of study participants.</p