Submitted Successfully!
To reward your contribution, here is a gift for you: A free trial for our video production service.
Thank you for your contribution! You can also upload a video entry or images related to this topic.
Version Summary Created by Modification Content Size Created at Operation
1
Pan Yan
 -- 2626 2023-02-23 04:25:26 |
2 update references and layout
Rita Xu
 Meta information modification 2626 2023-02-23 04:33:21 |

Video Upload Options

We provide professional Video Production Services to translate complex research into visually appealing presentations. Would you like to try it?
No, upload directly Yes

Confirm

Are you sure to Delete?
Yes No
Cite
If you have any further questions, please contact Encyclopedia Editorial Office.
Shang, T.; Fang, C.M.; Ong, C.E.; Pan, Y. Cytochrome P450 in Escherichia coli. Encyclopedia. Available online: https://encyclopedia.pub/entry/41571 (accessed on 26 December 2024).
Shang T, Fang CM, Ong CE, Pan Y. Cytochrome P450 in Escherichia coli. Encyclopedia. Available at: https://encyclopedia.pub/entry/41571. Accessed December 26, 2024.
Shang, Tao, Chee Mun Fang, Chin Eng Ong, Yan Pan. "Cytochrome P450 in Escherichia coli" Encyclopedia, https://encyclopedia.pub/entry/41571 (accessed December 26, 2024).
Shang, T., Fang, C.M., Ong, C.E., & Pan, Y. (2023, February 23). Cytochrome P450 in Escherichia coli. In Encyclopedia. https://encyclopedia.pub/entry/41571
Shang, Tao, et al. "Cytochrome P450 in Escherichia coli." Encyclopedia. Web. 23 February, 2023.
Cytochrome P450 in Escherichia coli
Edit
This entry is adapted from the peer-reviewed paper 10.3390/biotech12010017

Cytochrome P450 (CYP) enzymes play important roles in metabolising endogenous and xenobiotic substances. Characterisations of human CYP proteins have been advanced with the rapid development of molecular technology that allows heterologous expression of human CYPs. Among several hosts, bacteria systems such as Escherichia coli (E. coli) have been widely used thanks to their ease of use, high level of protein yields, and affordable maintenance costs.

human cytochrome P450 heterologous expression Escherichia coli

1. Introduction

Cytochrome P450 (CYP) enzymes are a group of membrane-bound hemoproteins responsible for the synthesis of a great number of endogenous compounds including steroid hormones, bile acids, fatty acids, and eicosanoids [1][2][3]. CYPs are also major phase I metabolizing enzymes, bio-transforming xenobiotics such as drugs and carcinogens, in the body [4][5]. In humans, the CYP families 1, 2, and 3 contribute significantly to xenobiotic metabolism, while other CYPs are mainly involved in endogenous biotransformation [6]. Unlike prokaryotic CYPs, which are soluble, mammalian CYPs are integral membrane proteins found in the endoplasmic reticulum or mitochondria [7]. Characterisations of the structure–function relationships for CYP enzymes have been impeded by the challenges of purifying these insoluble CYPs from human tissues with sufficient quantity and activity [8][9]. Moreover, with the advanced development of whole-genome sequencing technologies, a large number of CYP genomic variations have been identified [10]. CYP polymorphisms, in particular, CYP2C9, CYP2C19, and CYP2D6, account for the most commonly seen variations in phase I drug metabolism clinically [11]. Nevertheless, the low frequencies of CYP variants have limited the evaluations of their impact on the pharmacokinetics of clinical drugs [12].
The heterologous expression systems provide an alternative opportunity to obtain individual CYP isoforms and their variants in evaluating the enzyme activities or in analysing protein structures under reproducible conditions [13]. Thus far, several in vitro expression systems, including mammalian cells, baculoviruses, yeast, and bacteria cells, have been documented for applications in characterising CYP enzymes [14]. Mammalian cells such as the African green monkey kidney-derived cells COS-1 and the human embryonic kidney cells HEK293 have been employed in expressing recombinant human CYP enzymes [15][16]. The advantages of the mammalian cell systems include no requirement for cDNA modifications, as well as adequate levels of endogenous NADPH-CYP oxidoreductase (OxR) and cytochrome b5 to support electron transport and CYP catalytic activities [17]. However, employment of mammalian cells is often associated with high technical demand and a long duration of culture [18]. Besides, the CYP expression levels in mammalian cell cultures are usually low, which is unsuitable to study CYP variants, in particular, with low enzyme activity [14]. Baculovirus systems employ insect cells to express recombinant human CYPs, which can achieve high levels of expression [19]. Nevertheless, the technical demand and cost for insect cell cultures are high. The baculovirus systems also require the co-expression of OxR as insect cell lines are unable to express sufficient levels of OxR [17]. Yeasts such as Saccharomyces cerevisiae and Schizosaccharomyces pombe are useful in expressing human recombinant CYP [20][21]. The advantages of using yeast cells are low cost for maintenance, ease of culture, and a relatively high yield of CYP proteins. Moreover, the protein expression and post-translational modification processes are similar to those of higher eukaryotes, hence modifications of cDNA are usually not required [17]. Despite that yeasts contain endogenous OxR, the activity and quantity may be insufficient to fully support CYP enzyme activities, thus exogenous OxR may be essential [22]. Bacterial cells such as Escherichia coli (E. coli) demonstrate several advantages when being used as a heterologous system for human CYP expression. Culturing bacterial cells involves minimal maintenance cost as well as easier and faster cultivation. The recombinant CYP expression levels in bacteria are usually higher compared with those in yeast cells [23]. On the other hand, as human CYPs are membrane-bound, their expression in bacteria systems would require N-terminal modifications of the CYP cDNA to achieve optimal protein expression, conserve ideal folding, and maintain native biological functions [7][24].

2. Selections of Expression Vectors and E. coli Strains

The successful expression of CYP protein in bacteria is also influenced by the choice of plasmid vectors and E. coli strains (see Table 1).
The most commonly employed CYP expression plasmid vector in E. coli is pCWori+. It was initially developed by F.W. Dahlquist and is not commercially available [23]. The overall structure of pCWori+ has been illustrated previously [25]. Essentially, it contains two tac promoters upstream of the Nde I restriction enzyme digestion site coincident with the ATG codon (start codon). Only one tac promoter (the one upstream of the polylinker site) is used, which is recognised by E. coli RNA polymerase. Upon the addition of Isopropyl β-D-1-thiogalactopyranoside (IPTG), the protein expression output is proportional to the amount of IPTG, which allows the expression of the precise level of CYP [23]. Additionally, it contains one trpA (a strong transcription terminator), the β-lactamase gene (conferring ampicillin resistance), and the lacIq gene that encodes the Lac repressor (prevents any transcription initiated from the tac promoters without adding inducing agents) [25]. In general, the target CYP cDNA (native or modified) is introduced between the ATG start codon (contained within the Nde I site) and another restriction enzyme site, which is usually carried out by polymerase chain reaction (PCR) mutagenesis [26].
The recombinant vector was used in the transformation of various E. coli strains to produce recombinant human CYP proteins. Among them, DH5α [8][9][24][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48] and JM109 [24][26][29][49][50][51][52][53][54] strains are the most commonly used, while MV1304 [7][55][56][57], XL-1 blue [58], and TOPP [59][60] have also been used. It is important to note that the E. coli strain selection can impact CYP expression levels. It was evidenced that CYP2C10 was not detectable in JM109 cells, but expressed in DH5α cells [24]. Nevertheless, no genetic markers were identified in these strains, showing a significant correlation with the capability of producing high levels of recombinant CYP proteins [25]. It is suggested to evaluate these common E. coli strains for their ability to express a particular recombinant CYP at the beginning of the study.
Table 1. External contributing factors for selected human CYP expression in E. coli.
CYP Expression Vector E. coli Strain LB to TB Ratio OD600 Temp (°C) Shaking Speed (rpm) Duration (hour) IPTG (mM) Δ-ALA (mM) Reference
2E1 pCWori+ MV1304 1:90 0.5–0.8 N/A N/A 4 1 N/A [7]
17A1 pCWori+ JM109 N/A 0.4–0.8 28 N/A 48 1 N/A [26]
1A2 pCWori+ DH5α 1:100 N/A 30 125 72 1 N/A [38]
3A4 pCWori+ DH5α 1:100 N/A 32 N/A 24 1 N/A [31]
2E1&2B4 pJL MV1304 N/A 1.0 N/A N/A 4 1 N/A [55]
1A1 pCWori+ DH5α 1:100 N/A 30 Vigorous 48 N/A N/A [32]
2E1 pCWori+ DH5α 1:100 N/A 30 Vigorous 48 1 N/A [40]
1A2 pCWori+ DH5α or JM109 1:100 N/A 30 Vigorous 48 1 N/A [29]
7A1 pJL TOPP3 N/A 0.4–0.6 30 150 15–18 1 0.2 [59]
17A1-OxR pCWori+ DH5α 1:111 N/A 27 125 72 1 N/A [41]
3A5 pCWori+ DH5α 1:100 N/A 30 220 24 1 N/A [42]
2D6 pDS9 JM109 1:10 to 40 0.7–0.9 23 100 48 5 µM 0.5–1 [50]
2D6 pCWori+ DH5α N/A N/A 30 200 43 1.5 0.5 [30]
2E1-OxR pJL2 XL Blue N/A 0.8 26 100 60 1 N/A [58]
1A1-OxR pCWori+ DH5α 1:100 N/A 28 125 48 1 0.5 [43]
27A1 pTrc99A TOPP3 1:100 N/A 29 210 48 0.5 0.5 [60]
1A2-OxR pCWori+ DH5α 1:100 N/A 28 125 48 1 0.5 [61]
1B1-OxR pCWori+ DH5α 1:100 N/A 30 200 24 1 0.5 [33]
3A5 pCWori+ DH5α 1:100 0.3 30 160 28 0.1 1 [44]
2A6 pCWori+ DH5α 1:100 N/A 32 200 40 1 0.5 [34]
2B6 pCWori+ MV1304 N/A N/A 28 200 40–48 1 0.5 [56]
2D6-OxR pCWori+ DH5α 1:100 0.6–1.0 26 190 40–48 1 0.5 [28]
1A2-HDJ-1 pCWori+ DH5α N/A 0.4–0.5 37 N/A 24 1 1.5 [27]
2B6-GroES/EL pCWori+ JM109 1:100 N/A 30 160 72 1 0.5 [49]
27C1 pCWori+ JM109 1:100 N/A 27 200 48 1 0.5 [52]
4X1 pCWori+ DH5α 1:100 0.5 28 190 17–21 1 0.5 [35]
2S1 pBdtacHR LMG194 1:400 N/A 30 115 24–36 0.5 0.5 [62]
1A1-OxR pCWori+ DH5α 1:1000 0.5–0.7 30 200 24 1 0.5 [8]
2C10 &2C9 pCWori+ DH5α and JM109 1:100 N/A 30 Vigorous 24 1 N/A [24]
4A11 pCWori+ DH5α N/A 0.5 28 200 48 1 0.5 [46]
2J2 pCWori+ DH5α N/A N/A 28 N/A 48 1 0.5 [47]
4B1 pCWori+ DH5α N/A 0.4 27 120 48 1 0.5 [48]
39A1-GroEL/ES pCW-LIC C41 N/A 0.6 26 110 48 0.5 0.5 [63]
2J2-GroEL/ES pCWori+ DH5α 1:100 0.4–0.6 28 180 48 1 0.5 [9]

3. Bacteria Culture and Protein Expression Conditions

The typical bacteria culture and protein expression start with the initial culture of transformed E. coli strain in LB media supplemented with ampicillin (50–100 µg/mL) overnight at 37 °C (the optimal growth temperature for E. coli), followed with growing in Terrific Broth (TB) media containing ampicillin for an extended number of hours. The protein expression is subsequently induced by adding an inducing agent such as IPTG [31]. Factors involved in this process that may affect the yield of CYP protein expression include the ratio of LB to TB, OD600 readings upon initiation of protein expression, temperature, shaking speed, expression duration, concentrations of IPTG, with or without δ-aminolevulinic acid (δ-ALA), and other more specific conditions for a particular CYP isoform (see Table 1).
TB is a type of phosphate-buffered media that maintains a neutral pH level and comprises readily utilisable carbon sources [25]. The LB culture-to-TB culture ratio is usually maintained at 1:100 (e.g., 10 mL of LB culture to 1 L of TB) [31][38]. The TB media is often supplemented with trace elements to maintain CYP enzyme stability. Different studies applied different trace element compositions. As reported by Ahn and colleagues, trace elements expressing CYP1A2 in E. coli included 50 µM FeCl3, 1 mM MgCl2, and 2.5 mM (NH4)2SO4 [27]. It is common for 1 mM thiamine (also known as vitamin B1) to be added to the TB culture media to ensure rapid E. coli growth [64]. The typical OD600 values of 0.4 to 0.8 representing the mid-exponential bacterial growth phase were mostly used prior to induction [7][28]. Arabinose was required to induce the chaperon GroES-GroEL [9][35][62].
IPTG is a compound that mimics the molecular structure of allolactose that triggers the transcription of lac operon in E. coli. Hence, IPTG is used for protein expression induction where the gene expression is controlled by the lac operator, including pCWori+, the most commonly used vector for heterologous CYP protein expression in E. coli [25]. The majority of the studies employed 1 mM IPTG to induce CYP expression in E. coli cells, while exceptions were found in the expressions of CYP2D6 (1.5 mM IPTG) [30], CYP3A5 (0.1 mM IPTG) [44], CYP2S1, and CYP39A1 (0.5 mM) [62][63]. Δ-ALA, a well-known heme precursor, is involved in the pathway of protoporphyrin IX synthesis, and thus heme synthesis [65]. E. coli cells are able to produce heme-containing proteins with their endogenous heme biosynthesis system. The current results show that, although not an exclusive requirement for maximal production of all human CYP proteins in E. coli, the supplementation of δ-ALA could enhance the expression dramatically [25]. δ-ALA is readily taken up by E. coli cells, followed by heme synthesis catalysed by bacterial enzymes, which is subsequently inserted into the recombinant CYP polypeptide to form an enzymatically active protein [66]. The most commonly used final concentration of δ-ALA added before induction is 0.5 mM, with exceptions such as 1 mM for CYP3A5 [44] and 1.5 mM for CYP1A2 [27]. The addition of other chemicals to expression media was more specific to one or a group of CYP proteins. 4-methyl pyrazole, an inhibitor of CYP2E1 with high affinity, was added to the expression culture to stabilise the protein [7][58][67]. Bactopeptone was seeded in a TB medium to enhance cell growth in several studies [24][27][43][56].
The employment of 37 °C for protein expression usually results in recombinant CYP accumulating as inclusion bodies. A lower expression temperature has been shown to produce more stable proteins without aggregation [68]. Nevertheless, expression temperatures below 25 °C lead to a dramatic drop in the expression level [23]. The optimal expression temperature during protein induction is often within a rather narrow range, and thus sensitive to drastic fluctuations in the temperature of the incubator. The typical induction temperature is not higher than 30 °C (mostly 28–30 °C). Certain human CYP proteins can be expressed with higher yields under higher temperatures, such as CYP2A6, CYP2E1, and CYP1A2, which were expressed at a comparable level and activities at 37 °C [27][69]. Moreover, the shaking speed and length of incubation during induction may also influence the optimal expression levels. The culture media in flasks shaken vigorously at 100–200 rpm were routinely performed to obtain optimal yields [48][56]. During the induction phase, the incubation usually lasts for 24–72 h. For instance, Bui and Hankinson reported that the growth of E. coli at 30 °C for 24 h provided the best expression conditions for a recombinant CYP2S1 [62].

4. Membrane Isolation

At the end of protein expression, bacterial cells are harvested by centrifugation, followed by membrane isolation prior to purification. The general steps of membrane isolation include suspension of harvested cells, lysis of cells, removal of cell debris, and membrane fraction sedimentation by ultra-centrifugation. Different studies applied different protocols in terms of suspension buffer, lysis of cell methods (by a high-pressure homogenizer, lysozyme, and ultrasonic energy), choice of a protease inhibitor, and collection of membrane fraction sedimentation.
The harvested cells were usually suspended in phosphate buffers [57][63] or tris acetate buffers [29][31] with a pH range of 7.4–7.8 containing additional common compositions such as ethylenediamine tetraacetic acid (EDTA), sucrose, dithiothreitol (DTT), and glycerol. All of the steps were carried out at 4 °C. Both buffers functioned equally well in suspending bacterial cells expressing various recombinant human CYP proteins. Bacteria cells were suspended in a concentrated sucrose solution supplemented with EDTA, which were subsequently re-suspended in cold water. Under this condition, the bacteria cells shrink as a result of the high osmotic strength of the sucrose solution. EDTA plays a role in releasing lipopolysaccharide (LPS) from the cell envelope of bacterial cells, hence increasing the permeability of the outer membrane. Cold water leads to the rapid enlargement of cell size, resulting in the release of periplasmic proteins. This technique for the recovery of recombinant protein from E. coli is known as an osmotic shock [70]. Serious challenges have occurred in preserving protein stability and activity in biological applications as they are just marginally stable [71]. DTT is one of the protein reductants responsible for breaking down protein disulfide bridges and stabilizing enzymes [72]. Moreover, the most widely employed co-solvents for protein stabilization are polyols and, among polyols, glycerol is one of the most commonly used to stabilize and avoid aggregation of the protein [73][74].
Cell lysis can be defined as the destruction of the outer boundary or cell membrane to release inter-cellular materials. Cell lysis methods can be classified into mechanical (such as high-pressure homogenizer and bead mill) and non-mechanical approaches (including physical and chemical disruption) [75]. For the lysis of E. coli cells to obtain expressed human CYP proteins, mechanical approaches that use high-pressure homogenizer and non-mechanical techniques employing ultrasonic cavitation and enzymatic cell lysis were often recorded. A high-pressure homogenizer disrupts the membrane of cells by forcing them through an orifice valve [7][63]. Additionally, lysozyme is usually added to the suspended cell solution and incubated on ice or at 4 °C with stirring or shaking for 30 min [8][36]. Lysozyme is specific towards bacterial cells and reacts with the peptidoglycan layer, leading to the breaking of the glycosidic bond in the bacterial cell wall [76]. Ultrasonic cavitation is routinely applied in laboratories to disrupt cells. Ultrasound waves generate ultrasonic energy, which is transferred into the liquid solution and results in negative pressure. Once the negative pressure is lower than the vapour pressure of the liquid, vapour-filled bubbles are formed in the liquid solution. Then, when the bubbles grow to the size at which the ultrasonic energy is insufficient to maintain the vapour inside, they collapse and release a large amount of mechanical energy in the form of a shock wave, leading to cell rupture [77]. One of the disadvantages of ultrasonic cavitation is the generation of a large amount of heat, which may degrade enzymes [75]. During the lysis of E. coli, cells to isolate recombinant CYP proteins, a few rounds of ultrasonic treatment along with intervals on the ice were carried out in an ice bath to maintain cold conditions [24][26].
Upon lysis of cells, proteases are also released and their digestive functions are triggered, which can degrade isolated CYP enzyme proteins. Hence, the addition of protease inhibitors is required to preserve protein from imminent natural degradation. The majority of the proteases found in E. coli cells belong to the class of the serine protease group. Among the many classes of protease inhibitors, phenylmethylsulfonyl fluoride (PMSF) that inhibits serine protease irreversibly by deactivating the serine hydroxyl group is the most commonly used [78]. More recently, protease inhibitor cocktails comprising a mixture of several inhibitor compounds are more preferred in targeting a wide range of proteases that degrade enzymes via different mechanisms [28][54].

References

  1. Dong, L.; Wang, H.; Chen, K.; Li, Y. Biomedicine & Pharmacotherapy Roles of hydroxyeicosatetraenoic acids in diabetes (HETEs and diabetes). Biomed. Pharmacother. 2022, 156, 113981.
  2. Pikuleva, I.A.; Cartier, N. Cholesterol Hydroxylating Cytochrome P450 46A1: From Mechanisms of Action to Clinical Applications. Front. Aging Neurosci. 2021, 13, 1–17.
  3. Fujino, C.; Sanoh, S.; Katsura, T. Recent Advances in the Understanding of Nuclear Receptors- and Drug-Metabolizing Enzymes-Mediated Inter-Individual Differences Variation in Expression of Cytochrome P450 3A Isoforms and Toxicological Effects: Endo- and Exogenous Substances as Regulatory. Biol. Pharm. Bull. 2021, 44, 1617–1634.
  4. Perepechaeva, M.L.; Grishanova, A.Y. The Role of CYP3A in Health and Disease. Biomedicines 2022, 10, 2686.
  5. Luo, B.I.N.; Yan, D.; Yan, H.; Yuan, J. Cytochrome P450: Implications for human breast cancer (Review). Oncol. Lett. 2021, 22, 548.
  6. Gonzalez, F.J.; Nebert, D.W. Evolution of the P450 gene superfamily: Animal-plant “warfare”, molecular drive and human genetic differences in drug oxidation. Trends Genet. 1990, 6, 182–186.
  7. Larson, J.R.; Coon, M.J.; Porter, T.D. Purification and properties of a shortened form of cytochrome P-450 2E1: Deletion of the NH2-terminal membrane-insertion signal peptide does not alter the catalytic activities. Proc. Natl. Acad. Sci. USA 1991, 88, 9141–9145.
  8. Lee, S.H.; Kang, S.; Dong, M.S.; Park, J.D.; Park, J.; Rhee, S.; Ryu, D.Y. Characterization of the Ala62Pro polymorphic variant of human cytochrome P450 1A1 using recombinant protein expression. Toxicol. Appl. Pharmacol. 2015, 285, 159–169.
  9. Jeong, D.; Park, H.; Lim, Y.; Lee, Y.; Kim, V.; Cho, M.; Kim, D. Drug Metabolism and Pharmacokinetics Terfenadine metabolism of human cytochrome P450 2J2 containing genetic variations (G312R, P351L and P115L). Drug Metab. Pharmacokinet. 2018, 33, 61–66.
  10. Van Der Wouden, C.H.; Van Rhenen, M.H.; Jama, W.O.M.; Ingelman-sundberg, M.; Lauschke, V.M.; Konta, L.; Schwab, M.; Swen, J.J.; Guchelaar, H. Development of the PGx-Passport: A Panel of Actionable Germline Genetic Variants for Emptive Pharmacogenetic Testing. Clin. Pharmacokinet. 2019, 106, 866–873.
  11. Zhou, S.; Di, Y.M.; Chan, E.; Du, Y.; Chow, V.D.; Xue, C.C.; Lai, X.; Wang, J.; Li, C.G.; Tian, M.; et al. Clinical Pharmacogenetics and Potential Application in Personalized Medicine. Curr. Drug Metab. 2008, 9, 738–784.
  12. Fujikura, K.; Ingelman-sundberg, M.; Lauschke, V.M. Genetic variation in the human cytochrome P450 supergene family. Pharmacogenet. Genom. 2015, 25, 584–594.
  13. Kumondai, M.; Hishinuma, E.; Marie, E.; Rico, G.; Ito, A. Heterologous expression of high-activity cytochrome P450 in mammalian cells. Sci. Rep. 2020, 10, 1–13.
  14. Iratsuka, M.H. Review In Vitro Assessment of the Allelic Variants of Cytochrome P450. Drug Metab. Pharmacokinet. 2012, 27, 68–84.
  15. Taimi, M.; Helvig, C.; Wisniewski, J.; Ramshaw, H.; White, J.; Amad, M.; Korczak, B.; Petkovich, M. A Novel Human Cytochrome P450, CYP26C1, Involved in Metabolism of 9-cis and All-trans Isomers of Retinoic Acid. J. Biol. Chem. 2004, 279, 77–85.
  16. Sonawane, V.R.; Siddique, M.U.M.; Gatchie, L.; Williams, I.S.; Bharate, S.B.; Jayaprakash, V.; Sinha, B.N.; Chaudhuri, B. CYP enzymes, expressed within live human suspension cells, are superior to widely-used microsomal enzymes in identifying potent CYP1A1/CYP1B1 inhibitors: Identification of quinazolinones as CYP1A1/CYP1B1 inhibitors that efficiently reverse BP toxicity. Eur. J. Pharm. Sci. 2019, 131, 177–194.
  17. Schroer, K.; Kittelmann, M.; Lütz, S. Recombinant human cytochrome P450 monooxygenases for drug metabolite synthesis. Biotechnol. Bioeng. 2010, 106, 699–706.
  18. Kusano, K.; Sakaguchi, M.; Kagawa, N.; Waterman, M.R.; Omura, T. Microsomal P450s use specific proline-rich sequences for efficient folding, but not for maintenance of the folded structure. J. Biochem. 2001, 129, 259–269.
  19. Miyauchi, Y.; Kimura, A.; Sawai, M.; Fujimoto, K.; Hirota, Y.; Tanaka, Y.; Takechi, S.; Mackenzie, P.I.; Ishii, Y. Use of a Baculovirus-Mammalian Cell Expression-System for Expression of Drug-Metabolizing Enzymes: Optimization of Infection With a Focus on Cytochrome P450 3A4. Front. Pharmacol. 2022, 13, 832931.
  20. Imaoka, S.; Yamada, T.; Hiroi, T.; Hayashi, K.; Sakaki, T.; Yabusaki, Y.; Funae, Y. Multiple forms of human P450 expressed in Saccharomyces cerevisiae systematic characterization and comparison with those of the rat. Biochem. Pharmacol. 1996, 51, 1041–1050.
  21. Yasumori, T. Expression of a Human Cytochrome P450 Form in Schizosaccharomyces pombe: Comparison with Expression in Saccharomyces cerevisiae. In Foreign Gene Expression in Fission Yeast: Schizosaccharomyces pombe; Springer: Berlin/Heidelberg, Germany, 1997; pp. 111–121.
  22. Cheng, J.; Wan, D.F.; Gu, J.R.; Gong, Y.; Yang, S.L.; Hao, D.C.; Yang, L. Establishment of a yeast system that stably expresses human cytochrome P450 reductase: Application for the study of drug metabolism of cytochrome P450s in vitro. Protein Expr. Purif. 2006, 47, 467–476.
  23. Zelasko, S.; Palaria, A.; Das, A. Optimizations to achieve high-level expression of cytochrome P450 proteins using Escherichia coli expression systems. Protein Expr. Purif. 2013, 92, 77–87.
  24. Sandhu, P.; Baba, T.; Guengerich, F.P. Expression of modified cytochrome P450 2C10 (2C9) in Escherichia Coli, purification, and reconstitution of catalytic activity. Arch. Biochem. Biophys. 1993, 306, 443–450.
  25. Barnes, H.J. Maximizing Expression P450s of Eukaryotic. In Methods in Enzyymology; Academic Press: Cambridge, MA, USA, 1996; Volume 272, pp. 3–14.
  26. Barnes, H.J.; Arlotto, M.P.; Waterman, M.R. Expression and enzymatic activity of recombinant cytochrome P450 17α-hydroxylase in Escherichia coli. Proc. Natl. Acad. Sci. USA 1991, 88, 5597–5601.
  27. Ahn, T.; Yang, S.; Yun, C.H. High-level expression of human cytochrome P450 1A2 by co-expression with human molecular chaperone HDJ-1(Hsp40). Protein Expr. Purif. 2004, 36, 48–52.
  28. Deeni, Y.Y.; Paine, M.J.I.; Ayrton, A.D.; Clarke, S.E.; Chenery, R.; Wolf, C.R. Expression, purification, and biochemical characterization of a human cytochrome P450 CYP2D6-NADPH cytochrome P450 reductase fusion protein. Arch. Biochem. Biophys. 2001, 396, 16–24.
  29. Sandhu, P.; Guo, Z.; Baba, T.; Martin, M.V.; Tukey, R.H.; Guengerich, F.P. Expression of Modified Human Cytochrome P450 1A2 in Escherichia coli: Stabilization, Purification, Spectral Characterization, and Catalytic Activities of the Enzyme. Arch. Biochem. Biophys. 1994, 309, 168–177.
  30. Gillam, E.M.J.; Zuyu, G.; Martin, M. V Expression of cytochrome P450 2D6 in Escherichia coli, purification, and spectral and catalytic characterization. Arch. Biochem. Biophys. 1995, 319, 540–550.
  31. Gillam, E.M.J.; Baba, T.; Kim, B.R.; Ohmori, S.; Guengerich, F.P. Expression of Modified Human Cytochrome P450 3A4 in Escherichia coli and Purification and Reconstitution of the Enzyme. Arch. Biochem. Biophys. 1993, 305, 123–131.
  32. Guo, Z.; Gillam, E.M.J.; Ohmori, S.; Tukey, R.H.; Peter Guengerich, F. Expression of modified human cytochrome P450 1A1 in Escherichia coli: Effects of 5’ substitution, stabilization, purification, spectral characterization, and catalytic properties. Arch. Biochem. Biophys. 1994, 312, 436–446.
  33. Shimada, T.; Wunsch, R.M.; Hanna, I.H.; Sutter, T.R.; Guengerich, F.P.; Gillam, E.M.J. Recombinant human cytochrome P450 1B1 expression in Escherichia coli. Arch. Biochem. Biophys. 1998, 357, 111–120.
  34. Souček, P. Expression of cytochrome P450 2A6 in Escherichia coli: Purification, spectral and catalytic characterization, and preparation of polyclonal antibodies. Arch. Biochem. Biophys. 1999, 370, 190–200.
  35. Stark, K.; Dostalek, M.; Guengerich, F.P. Expression and purification of orphan cytochrome P450 4X1 and oxidation of anandamide. FEBS J. 2008, 275, 3706–3717.
  36. Pan, Y.; Abd-Rashid, B.A.; Ismail, Z.; Ismail, R.; Mak, J.W.; Ong, C.E. Heterologous Expression of Human Cytochromes P450 2D6 and CYP3A4 in Escherichia coli and Their Functional Characterization. Protein J. 2011, 30, 581–591.
  37. Wu, Z.L.; Sohl, C.D.; Shimada, T.; Guengerich, F.P. Recombinant enzymes overexpressed in bacteria show broad catalytic specificity of human cytochrome P450 2W1 and limited activity of human cytochrome P450 2S1. Mol. Pharmacol. 2006, 69, 2007–2014.
  38. Fisher, C.W.; Caudle, D.L.; Martin-Wixtrom, C.; Quattrochi, L.C.; Tukey, R.H.; Waterman, M.R.; Estabrook, R.W. High-level expression of functional human cytochrome P450 1A2 in Escherichia coli. FASEB J. 1992, 6, 759–764.
  39. Shet, M.S.; Fisher, C.W.; Holmans, P.L.; Estabrook, R.W. Human cytochrome P450 3A4: Enzymatic properties of a purified recombinant fusion protein containing NADPH-P450 reductase. Proc. Natl. Acad. Sci. USA 1993, 90, 11748–11752.
  40. Gillam, E.M.J.; Guo, Z.; Peter Guengerich, F. Expression of modified human cytochrome P450 2E1 in Escherichia coli, purification, and spectral and catalytic properties. Arch. Biochem. Biophys. 1994, 312, 59–66.
  41. Shet, M.S.; Fisher, C.W.; Arlotto, M.P.; Shackleton, C.H.L.; Holmans, P.L.; Martin-Wixtrom, C.A.; Saeki, Y.; Estabrook, R.W. Purification and enzymatic properties of a recombinant fusion protein expressed in Escherichia coli containing the domains of bovine P450 17A and rat NADPH-P450 reductase. Arch. Biochem. Biophys. 1994, 311, 402–417.
  42. Gillam, E.M.J.; Guo, Z.Y.; Ueng, Y.F.; Yamazaki, H.; Cock, I.; Reilly, P.E.B.; Hooper, W.D.; Guengerich, F.P. Expression of cytochrome-p450-3a5 in escherichia coli: Effects of 5′ modification, purification, spectral characterization, reconstitution conditions, and catalytic activities. Arch. Biochem. Biophys. 1995, 317, 374–384.
  43. Chun, Y.J.; Shimada, T.; Guengerich, F.P. Construction of a human cytochrome P450 1A1:Rat NADPH-cytochrome P450 reductase fusion protein cDNA and expression in Escherichia coli, purification, and catalytic properties of the enzyme in bacterial cells and after purification. Arch. Biochem. Biophys. 1996, 330, 48–58.
  44. Yamakoshi, Y.; Kishimoto, T.; Sugimura, K.; Kawashima, H. Human prostate CYP3A5: Identification of a unique 5’-untranslated sequence and characterization of purified recombinant protein. Biochem. Biophys. Res. Commun. 1999, 260, 676–681.
  45. Shimada, T.; Tsumura, F.; Gillam, E.M.J.; Guengerich, F.P.; Inoue, K. Roles of NADPH-P450 reductase in the O-deethylation of 7-ethoxycoumarin by recombinant human cytochrome P450 1B1 variants in Escherichia coli. Protein Expr. Purif. 2000, 20, 73–80.
  46. Choi, S.; Han, S.; Lee, H.; Chun, Y.; Kim, D. Evaluation of Luminescent P450 Analysis for Directed Evolution of Human CYP4A11. Biomol. Ther. 2013, 21, 487–492.
  47. Park, H.; Lim, Y.; Han, S.; Kim, D. Expression and Characterization of Truncated Recombinant Human Cytochrome P450 2J2. Toxicol. Res. 2014, 30, 33–38.
  48. Roellecke, K.; Jäger, V.D.; Gyurov, V.H.; Kowalski, J.P.; Mielke, S.; Rettie, A.E.; Hanenberg, H.; Wiek, C.; Girhard, M. Ligand characterization of CYP4B1 isoforms modified for high-level expression in Escherichia coli and HepG2 cells. Protein Eng. Des. Sel. 2017, 30, 207–218.
  49. Mitsuda, M.; Iwasaki, M. Improvement in the expression of CYP2B6 by co-expression with molecular chaperones GroES/EL in Escherichia coli. Protein Expr. Purif. 2006, 46, 401–405.
  50. Kempf, A.C.; Zanger, U.M.; Meyer, U.A. Truncated Human P450 2D6P: Expression in Escherichia coli, Ni2+-Chelate Affinity Purification, and Characterization of Solubility and Aggregation. Arch. Biochem. Biophys. 1995, 321, 277–288.
  51. Richardson, T.H.; Jung, F.; Griffin, K.J.; Wester, M.; Raucy, J.L.; Kemper, B.; Bornheim, L.M.; Hassett, C.; Omiecinski, C.J.; Johnson, E.F. A universal approach to the expression of human and rabbit cytochrome P450s of the 2C subfamily in Escherichia coli. Arch. Biochem. Biophys. 1995, 323, 87–96.
  52. Wu, Z.L.; Bartleson, C.J.; Ham, A.J.L.; Guengerich, F.P. Heterologous expression, purification, and properties of human cytochrome P450 27C1. Arch. Biochem. Biophys. 2006, 445, 138–146.
  53. Appiah-Opong, R.; Commandeur, J.N.M.; Axson, C.; Vermeulen, N.P.E. Interactions between cytochromes P450, glutathione S-transferases and Ghanaian medicinal plants. Food Chem. Toxicol. 2008, 46, 3598–3603.
  54. Milichovký, J.; Bárta, F.; Schmeiser, H.H.; Arlt, V.M.; Frei, E.; Stiborová, M.; Martínek, V. Active site mutations as a suitable tool contributing to explain a mechanism of aristolochic acid I nitroreduction by cytochromes P450 1A1, 1A2 and 1B1. Int. J. Mol. Sci. 2016, 17, 213.
  55. Pernecky, S.J.; Larson, J.R.; Philpot, R.M.; Coon, M.J. Expression of truncated forms of liver microsomal P450 cytochromes 2B4 and 2E1 in Escherichia coli: Influence of NH2-terminal region on localization in cytosol and membranes. Proc. Natl. Acad. Sci. USA 1993, 90, 2651–2655.
  56. Hanna, I.H.; Reed, J.R.; Guengerich, F.P.; Hollenberg, P.F. Expression of human cytochrome P450 2B6 in Escherichia coli: Characterization of catalytic activity and expression levels in human liver. Arch. Biochem. Biophys. 2000, 376, 206–216.
  57. Larson, J.R.; Coon, M.J.; Porter, T.D. Alcohol-inducible cytochrome P-450IIE1 lacking the hydrophobic NH2-terminal segment retains catalytic activity and is membrane-bound when expressed in Escherichia coli. J. Biol. Chem. 1991, 266, 7321–7324.
  58. Dong, J.; Porter, T.D. Coexpression of mammalian cytochrome P450 and reductase in Escherichia coli. Arch. Biochem. Biophys. 1996, 327, 254–259.
  59. Karam, W.G.; Chiang, J.Y.L. Expression and purification of human cholesterol 7α-hydroxylase in Escherichia coli. J. Lipid Res. 1994, 35, 1222–1231.
  60. Pikuleva, I.A.; Bjo, I.; Waterman, M.R. Expression, purification, and enzymatic properties of recombinant human cytochrome P450c27 (CYP27). Arch. Biochem. Biophys. 1997, 343, 123–130.
  61. Parikh, A.; Guengerich, F.P. Expression, purification, and characterization of a catalytically active human cytochrome P450 1A2:Rat NADPH-cytochrome P450 reductase fusion protein. Protein Expr. Purif. 1997, 9, 346–354.
  62. Bui, P.H.; Hankinson, O. Functional characterization of human cytochrome P450 2S1 using a synthetic gene-expressed protein in Escherichia coli. Mol. Pharmacol. 2009, 76, 1031–1043.
  63. Grabovec, I.P.; Smolskaya, S.V.; Baranovsky, A.V.; Zhabinskii, V.N.; Dichenko, Y.V.; Shabunya, P.S.; Usanov, S.A.; Strushkevich, N.V. Ligand-binding properties and catalytic activity of the purified human 24-hydroxycholesterol 7α-hydroxylase, CYP39A1. J. Steroid Biochem. Mol. Biol. 2019, 193, 105416.
  64. Elbing, K.; Llp, E.; Brent, R. Recipes and tools for culture of Escherichia coli. Curr. Protoc. Mol. Biol. 2019, 125, 1–19.
  65. Miura, M.; Ito, K.; Hayashi, M.; Nakajima, M.; Tanaka, T. The Effect of 5-Aminolevulinic Acid on Cytochrome P450-Mediated Prodrug Activation. PLoS ONE 2015, 10, e0131793.
  66. Yadav, R.; Scott, E.E. cro Endogenous insertion of non-native metalloporphyrins into human membrane cytochrome P450 enzymes. J. Biol. Chem. 2018, 293, 16623–16634.
  67. Koop, D.R. Inhibition of Ethanol-Inducible Cytochrome P450I I E I by. Chem. Res. Toxicol. 1990, 3, 377–383.
  68. San-miguel, T.; Pérez-bermúdez, P.; Gavidia, I. Production of soluble eukaryotic recombinant proteins in E. coli is favoured in early log-phase cultures induced at low temperature. Springerplus 2013, 2, 2–5.
  69. Yim, S.K.; Ahn, T.; Jung, H.C.; Pan, J.G.; Yun, C.H. Temperature effect on the functional expression of human cytochromes P450 2A6 and 2E1 in Escherichia coli. Arch. Pharm. Res. 2005, 28, 433–437.
  70. Chen, Y.; Chen, L.; Chen, S.; Chang, M.; Chen, T. A modified osmotic shock for periplasmic release of a recombinant creatinase from Escherichia coli. Biochem. Eng. J. 2004, 19, 211–215.
  71. Timasheff, S.N. By Weak Interactions with Water: How Do Solvents Affect These Processes? Annu. Rev. Biophys. Biomol. Struct. 1993, 22, 67–97.
  72. Fjelstrup, S.; Andersen, M.B.; Thomsen, J.; Wang, J.; Stougaard, M.; Pedersen, F.S.; Ho, Y.; Hede, M.S.; Knudsen, B.R. The Effects of Dithiothreitol on DNA. Sensors 2017, 17, 1201.
  73. Kaushik, J.K.; Bhat, R. Thermal Stability of Proteins in Aqueous Polyol Solutions: Role of the Surface Tension of Water in the Stabilizing Effect of Polyols. J. Phys. Chem. B 1998, 5647, 7058–7066.
  74. Vagenende, V.; Yap, M.G.S.; Trout, B.L. Mechanisms of Protein Stabilization and Prevention of Protein Aggregation by Glycerol. Biochemistry 2009, 48, 11084–11096.
  75. Islam, M.S.; Aryasomayajula, A.; Selvaganapathy, P.R. A Review on Macroscale and Microscale Cell Lysis Methods. Micromachines 2017, 8, 83.
  76. Acuña, J.M.B.; Hidalgo-dumont, C.; Pacheco, N.; Cabrera, A.; Poblete-castro, I. OPEN A novel programmable lysozyme- based lysis system in Pseudomonas putida for biopolymer production. Sci. Rep. 2017, 7, 1–11.
  77. Liu, Y.; Liu, X.; Cui, Y.; Yuan, W. Ultrasonics Sonochemistry Ultrasound for microalgal cell disruption and product extraction: A review. Ultrason. Sonochem. 2022, 87, 106054.
  78. Serine, A. Natural Product Communications Purification and Biochemical Characterization of. Nat. Prod. Commun. 2010, 5, 931–934.
More
© Text is available under the terms and conditions of the Creative Commons Attribution (CC BY) license; additional terms may apply. By using this site, you agree to the Terms and Conditions and Privacy Policy.
Upload a video for this entry
Information
Subjects: Biochemistry & Molecular Biology
Contributors MDPI registered users' name will be linked to their SciProfiles pages. To register with us, please refer to https://encyclopedia.pub/register :
Tao Shang
,
Chee Mun Fang
,
Chin Eng Ong
,
Yan Pan
View Times: 934
Revisions: 2 times (View History)
Update Date: 23 Feb 2023
Table of Contents
    1000/1000
    Hot Most Recent
    Video Production Service
    Feedback