Heterologous Expression of Recombinant Human Cytochrome P450 (CYP) in Escherichia coli: N-Terminal Modification, Expression, Isolation, Purification, and Reconstitution

被引:6
作者
Shang, Tao [1 ]
Fang, Chee Mun [1 ]
Ong, Chin Eng [2 ]
Pan, Yan [1 ]
机构
[1] Univ Nottingham Malaysia, Sch Pharm, Div Biomed Sci, Jalan Broga, Semenyih 43500, Malaysia
[2] Int Med Univ, Sch Pharm, Bukit Jalil, Kuala Lumpur 57000, Malaysia
来源
BIOTECH | 2023年 / 12卷 / 01期
关键词
human cytochrome P450; heterologous expression; Escherichia coli; purification; N-terminal modification; HIGH-LEVEL EXPRESSION; CATALYTIC-ACTIVITY; P450; REDUCTASE; SPECTRAL CHARACTERIZATION; ENZYMATIC-PROPERTIES; FUSION PROTEIN; CHOLESTEROL; 7-ALPHA-HYDROXYLASE; MICROSOMAL CYTOCHROME-P450; AFFINITY PURIFICATION; NONIONIC DETERGENT;
D O I
10.3390/biotech12010017
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
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. However, the levels of expression in E. coli reported in the literature sometimes differ significantly. This paper aims to review several contributing factors, including N-terminal modifications, co-expression with a chaperon, selections of vectors and E. coli strains, bacteria culture and protein expression conditions, bacteria membrane preparations, CYP protein solubilizations, CYP protein purifications, and reconstitution of CYP catalytic systems. The common factors that would most likely lead to high expression of CYPs were identified and summarised. Nevertheless, each factor may still require careful evaluation for individual CYP isoforms to achieve a maximal expression level and catalytic activity. Recombinant E. coli systems have been evidenced as a useful tool in obtaining the ideal level of human CYP proteins, which ultimately allows for subsequent characterisations of structures and functions.
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页数:18
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共 133 条
[1]  
Acuna J.M.B., 2017, SCI REP-UK, V7, P1
[2]   High-level expression of human cytochrome P450 1A2 by co-expression with human molecular chaperone HDJ-1 (Hsp40) [J].
Ahn, T ;
Yang, SY ;
Yun, CH .
PROTEIN EXPRESSION AND PURIFICATION, 2004, 36 (01) :48-52
[3]   Affinity purification of recombinant human cytochrome P450s 3A4 and 1A2 using mixed micelle systems [J].
Ahn, Taeho ;
Bae, Chun-Sik ;
Yun, Chul-Ho .
PROTEIN EXPRESSION AND PURIFICATION, 2014, 101 :37-41
[4]   Cytochrome b5 modulation of 17α hydroxylase and 17-20 lyase (CYP17) activities in steroidogenesis [J].
Akhtar, MK ;
Kelly, SL ;
Kaderbhai, MA .
JOURNAL OF ENDOCRINOLOGY, 2005, 187 (02) :267-274
[5]   Interactions between cytochromes P450, glutathione S-transferases and Ghanaian medicinal plants [J].
Appiah-Opong, Regina ;
Commandeur, Jan N. M. ;
Axson, Civianny ;
Vermeulen, Nico P. E. .
FOOD AND CHEMICAL TOXICOLOGY, 2008, 46 (12) :3598-3603
[6]  
Barnes Henry J., 1996, V272, P3
[7]   EXPRESSION AND ENZYMATIC-ACTIVITY OF RECOMBINANT CYTOCHROME-P450 17-ALPHA-HYDROXYLASE IN ESCHERICHIA-COLI [J].
BARNES, HJ ;
ARLOTTO, MP ;
WATERMAN, MR .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1991, 88 (13) :5597-5601
[8]   MEMBRANE TOPOLOGY OF THE MAMMALIAN P450-CYTOCHROMES [J].
BLACK, SD .
FASEB JOURNAL, 1992, 6 (02) :680-685
[9]   MECHANISM OF RATE CONTROL OF THE NADPH-DEPENDENT REDUCTION OF CYTOCHROME-P-450 BY LIPIDS IN RECONSTITUTED PHOSPHOLIPID-VESICLES [J].
BLANCK, J ;
SMETTAN, G ;
RISTAU, O ;
INGELMANSUNDBERG, M ;
RUCKPAUL, K .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1984, 144 (03) :509-513
[10]   Organization of NADPH-Cytochrome P450 Reductase and CYP1A2 in the Endoplasmic Reticulum-Microdomain Localization Affects Monooxygenase Function [J].
Brignac-Huber, Lauren ;
Reed, James R. ;
Backes, Wayne L. .
MOLECULAR PHARMACOLOGY, 2011, 79 (03) :549-557