Human cytochrome P450 3A5 enzyme and NADPH-cytochrome P450 oxidoreductase co-expression system

A cytochrome and reductase technology, applied in the field of genetic engineering, can solve the problems of complex reaction conditions and difficult separation of intermediate products

Inactive Publication Date: 2014-07-23
ZHONGSHAN PHARMASS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In addition, because the quantitative determination of metabolic activity requires a large number of metabolite standards, and the standard is now mainly synthesized by chemical methods, not only the reaction conditions are complicated, but also the separation of intermediate products is very difficult

Method used

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  • Human cytochrome P450 3A5 enzyme and NADPH-cytochrome P450 oxidoreductase co-expression system
  • Human cytochrome P450 3A5 enzyme and NADPH-cytochrome P450 oxidoreductase co-expression system
  • Human cytochrome P450 3A5 enzyme and NADPH-cytochrome P450 oxidoreductase co-expression system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Construction of human cytochrome P450 3A5 enzyme (CYP3A5) and NADPH-cytochrome P450 oxidoreductase (CYPOR) co-expression system

[0037] The specific steps of construction are as follows:

[0038] Digest the plasmid pBS-2A (containing the 2A peptide gene) with XbaI, recover the 3.0kb backbone fragment and the 130bp 2A connecting peptide respectively, dephosphorylate the 3.0kb backbone fragment, and reconnect to select the reverse clone, namely pBS-2A(- );

[0039] Insert the NADPH-cytochrome P450 oxidoreductase gene (CYPOR) between the BglII and Not I restriction sites of the vector pBS-2A(-) to obtain the vector pBS-2A-POR (see Figure 14 );

[0040] The human cytochrome P4503A5 enzyme gene was directional inserted between the BamH I and Xba I restriction sites of the vector pBS-2APOR to obtain the vector pBS-CYP3A5-2A-POR (see Figure 5-8 );

[0041] Digest pBS-CYP3A5-2A-POR with BamH I and Not I (results in Figure 9 ), insert the CYP3A5-2A-POR fragmen...

Embodiment 2

[0044] Example 2 Expression of Human Cytochrome P450 3A5 Enzyme in Pichia pastoris GS115

[0045] Positive clones were obtained after resistance and PCR screening, and the positive clones were subjected to methanol-induced expression for a certain period of time, and then the yeast cells were sonicated, and the protein expression was analyzed by conventional SDS-PAGE electrophoresis and Western Blot (see Figure 10 , 11 , 12), the results preliminarily proved that CYP3A5 was expressed in Pichia pastoris.

Embodiment 3

[0046] Example 3 Determination of Metabolic Midazolam Activity of Yeast Expressing Recombinant Human P4503A5 Enzyme

[0047] Homogenize the collected yeast cells with 0.1M phosphate buffer; centrifuge at 9,000g, 4°C for 20 minutes and at 100,000g, 4°C for 60 minutes to prepare microsomes by differential centrifugation; weigh in 0.1M phosphate buffer Suspended microsomes were precipitated, and the protein content was determined by the Bradford method, and the P450 content was determined by the carbon monoxide binding method; 0.45 mL of the mixture of the above-mentioned microsomes at an appropriate concentration and serially diluted midazolam was placed in a reaction tube, and kept in a 37°C water bath for 5 minute. Add 40 μL of 10 mM NADPH solution to start the reaction. After 6 minutes of reaction, take 0.1 mL of the reaction termination solution containing internal standard to terminate the reaction, mix well and place in ice water; centrifuge each of the above reaction tub...

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Abstract

The invention belongs to the field of gene engineering, and particularly relates to a human cytochrome P450 3A5 enzyme and an NADPH-cytochrome P450 oxidoreductase co-expression system. The co-expression system comprises host yeast, and a co-expression vector inserting into a P450 oxidoreductase gene and a human-source P450 3A5 gene in an oriented manner. The human cytochrome P450 3A5 enzyme and the NADPH-cytochrome P450 oxidoreductase co-expression system can be heterologously expressed in the yeast. In addition, a disadvantage of the low P450 oxidoreductase expression level in yeast cells is overcome. A transfected cell can co-express two proteins simultaneously and a large amount of metabolites can be prepared.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to the heterologous co-expression of human cytochrome P450 3A5 enzyme and cytochrome P450 oxidoreductase by using the method of genetic engineering with yeast as a host, and the preparation of products by metabolizing midazolam with human cytochrome P450 3A5 enzyme 1-Hydroxymidazolam method. Background technique [0002] Cytochrome P450 enzymes are important drug metabolism phase I enzymes, and more than 80% of drugs on the market are metabolized by cytochrome P450 enzymes. The 3A subfamily is the main member of the cytochrome P450 gene family, mainly including several members of 3A4, 3A5 and 3A7. Cytochrome P4503A5 enzyme can metabolize a variety of drugs such as midazolam (Midazolam), nifedipine (Nifedipine), testosterone, progesterone and so on. [0003] Traditional drug metabolism research is mainly based on animal experiments. In recent years, the preliminary dr...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/66C12P17/18
Inventor 戴仁科张伟黄黎珍邓继锋王珍玉谢水林
Owner ZHONGSHAN PHARMASS
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