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

Saluja D (2014) A Novel Methodology for Enhanced and Consistent Heterologous Expression of Unmodified Human Cytochrome P450 1B1 (CYP1B1). PLoS ONE 9(10): e110473. doi:10.1371/journal.pone.0110473 A Novel Methodology for Enhanced and Consistent Heterologous Expression of Unmodified Human Cytochrome P450 1B1 (CYP1B1) Muneeb A. Faiq 0 Mashook Ali 0 Tanuj Dada 0 Rima Dada 0 Daman Saluja 0 Ayyalusamy Ramamoorthy, University of Michigan, United States of America 0 1 Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences , Ansari Nagar, New Delhi , India , 2 Medical Biotechnology Laboratory, Dr. B. R. Ambedkar Centre for Biomedical Research, University of Delhi , North Campus, Delhi , India , 3 Laboratory for Molecular Reproduction and Genetics , Department of Anatomy, All India Institute of Medical Sciences , Ansari Nagar , India 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 b-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. - Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: The author(s) received no specific funding for this work but the institutional grant by Dr. B. R. Ambedkar Centre for Biomedical Research, University of Delhi to DS and funding by Indian Council of Medical Research, New Delhi to RD is acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. 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]. CYP1B1 gene is located on chromosomal locus 2p21-22 [3] comprising of 3 exons and 2 introns while both CYP1A1 and CYP1A2 are located on chromosome 15 and both are arranged in 7 exons and 6 introns [2]. As confirmed by the DNA hybridization studies, CYP1B1 is the only member of CYP1B subfamily [1,3]. Due to all the above reasons, the properties and functions of CYP1B1 cannot be predicted by the functional analysis of CYP1A1 and CYP1A2. Hence, 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. CYP1B1 is expressed in many tissues in the body including adipose tissue, eyes, brain, colon, embryo, heart, kidneys, lungs, muscle, pancreas, testes, thymus etc. [http://www.urogene.org/ pgdb/gene/107.html]. It is considered as a universal cancer marker [48] with implications in ovarian cancer [9], colorectal adenocarcinoma [10], acute lymphocytic leukemia, acute myeloid leukemia, esophageal carcinoma, lung cancers, lymphoma, rhabdomyosarcoma [6], prostate carcinoma [11] etc. In addition to this, CYP1B1 plays an important role in embryonic eye development [1214] and its mutations have been implicated in primary congenital glaucoma (PCG) [1517]. In our previous studies, we observed a high prevalence of CYP1B1 mutations in North Indian PCG patients and also reported 7 novel mutations [18,19]. We have also reviewed the molecular, biochemical, diagnostic, clinical and genetic aspects of CYP1B1 involvement in PCG [20,21]. Many investigators have reported enhanced expression of Nterminal modified CYP1B1 in E. coli [22,23]. However, such a protein may not be a pertinent representative for valid functional studies for obvious reasons. Change in any aminoacid in the native protein sequence may produce numerous (predictable/unpredictable) changes in the functional integrity of the translation product and may, as a consequence, render the functional studies invalid. Previous studies have also shown that N-terminal mutations are associated with many diseases, suggesting that this region is critical for the normal function of CYP1B1 [18,19]. A few examples of such already reported N-terminal mutations involved in pathogenesis of PCG are L24R, P54L, W57C, I60M, G61E, L77P and Y81N [24]. Hence, sufficient amount of the purified full length protein is required for carrying out the functional studies. Membrane proteins are difficult to express in prokaryotic expression systems for many reasons sufficiently elucidated by Orawan et al. (2012) [25] and many others [2628]. Cytochrome P450s are particularly difficult to express in procaryotic expression systems as there is often the possibility of formation of self hybridization derived secondary structures in mRNA transcripts [29]. This issue becomes (...truncated)