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Method used for increasing cyp3A5 expression enzyme metabolism activity

A metabolically active, double-expression technology, applied in the field of genetic engineering, can solve problems that consume a lot of manpower and material resources, and are complicated

Inactive Publication Date: 2018-07-06
广东睿谷生物科技有限公司
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  • Abstract
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  • Claims
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Problems solved by technology

[0007] At present, the production of CYP450 metabolites basically adopts chemical synthesis methods. The structures of the synthesized compounds need to be identified one by one, and the isomers produced in the chemical synthesis are separated, which is usually very complicated. The synthesis process takes several weeks and consumes a lot of manpower. material resources

Method used

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  • Method used for increasing cyp3A5 expression enzyme metabolism activity
  • Method used for increasing cyp3A5 expression enzyme metabolism activity
  • Method used for increasing cyp3A5 expression enzyme metabolism activity

Examples

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Embodiment 1

[0054] Example 1 Improvement of recombinant human cyp3A5 yeast His + Mut + Method for expressing enzyme metabolic activity of cyp3A5 in a bioreactor

[0055] Include the following steps:

[0056] 1. Construction of dual expression vector pPIC-CYP3A5-2A-POR

[0057] 1. Construction of pBS-2A(-) vector

[0058] Digest the plasmid pBS-2A (purchased from Guangzhou Funeng Gene Co., Ltd.) with XbaI, recover the 3.0kb PBS backbone fragment and the 130bp 2A connecting peptide fragment respectively, dephosphorylate the 3.0kb backbone fragment, and reconnect to select reverse cloning The vector pBS-2A(-) was obtained; the result is shown in Figure 1;

[0059] 2. Construction of vector pBS-2A-CYPOR

[0060] Insert the NADPH-cytochrome P450 oxidoreductase gene POR (purchased from Guangzhou Funeng Gene Co., Ltd.) between the Bgl II and Not I restriction sites of the vector pBS-2A(-) to obtain the vector pBS-2A -POR; the result is shown in Figure 2;

[0061] 3. Construction of vect...

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Abstract

The invention discloses a CYP3A5 bioreactor based on yeast, and a construction method thereof. According to the construction method, human derived CYP3A5 gene is subjected to sequence optimization based on the coding preference of yeast codons, gene engineering technology is adopted to realize cotransfection of human cyp3A5 obtained via sequence optimization and oxidoreductase DNA thereof into thegenome of yeast engineering bacterial strains, so that large amount of expression of cyp3A5 and NADPH in pichia yeast is realized, entering of drugs into cytoplasm through yeast cell walls with improved permeability is realized, selective metabolism on certain drugs in yeast fermentation culturing process is realized, the drug metabolism bioreactor based on gene engineering yeast is constructed,high efficiency specific production of drug metabolites through biosynthesis is realize, a reliable in vitro model is provided for pharmacology and toxicology research, and a novel research platform is provided for new drug exploitation of our country.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and more specifically, the invention relates to a method for detecting the metabolic activity of a recombinantly expressed enzyme CYP3A5 substrate. Background technique [0002] In the process of new drug research and development, the characteristics of drug absorption, distribution, metabolism, excretion and toxicity (ADME / Tox) play a crucial role in the ultimate success of a drug candidate. Cytochrome enzymes (cyp450) are very important phase I enzymes of drug metabolism. More than 85% of drugs currently on the market are metabolized by cyp450 in the human body. Traditional drug metabolism research is mainly based on in vivo experiments in experimental animals. In recent years, early drug metabolism research has entered the research stage of in vitro metabolism: that is, using recombinant cyp450 enzymes to express in heterologous expression systems, establishing an in vitro screeni...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N15/81C12N1/19C12N9/02C12R1/84
CPCC12N9/0071C12N15/815
Inventor 戴仁科王珍玉戴天明江伟凡郭佳音邓继峰张伟郭子钲
Owner 广东睿谷生物科技有限公司
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