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Method for improving in-vitro expression of cytochrome P450 enzyme family

A technology of in vitro expression and cytochrome, applied in botany equipment and methods, biochemical equipment and methods, plant gene improvement, etc., can solve problems such as difficult to meet research needs and low yield, and achieve good social benefits and considerable economic benefits benefit effect

Inactive Publication Date: 2011-06-15
NANJING MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, when using the classical method to prepare CYP450 and POR, it is found that the yield is very low, which is difficult to meet the research needs

Method used

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  • Method for improving in-vitro expression of cytochrome P450 enzyme family
  • Method for improving in-vitro expression of cytochrome P450 enzyme family
  • Method for improving in-vitro expression of cytochrome P450 enzyme family

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] 1. Construction and identification of recombinant transfer vector pFastBac1-CYP2A13 plasmid

[0034] pFastBac1-CYP2A13 is preserved in our laboratory. The Escherichia coli containing the recombinant plasmid was obtained by screening on the ampicillin resistance plate, and the DNA of the bacterial solution was extracted to obtain the plasmid DNA of the recombinant transfer vector. Validation of pFastBac1-CYP2A13 using double restriction digestion.

[0035] 2. Construction and identification of recombinant eukaryotic expression virus Ac-Bacmid-CYP2A13 DNA

[0036] The constructed pFastBac1-CYP 2A13 recombinant plasmid was transformed into DH10Bac Escherichia coli competent cells, and with the assistance of the helper plasmid Helper of DH10Bac Escherichia coli, transposoned by Tn7 transposon, each CYP2A13 gene fragment was inserted into the shuttle vector Bacmid, and Kanamycin, gentamicin and tetracycline three-antibody screening and blue-white screening to obtain recomb...

Embodiment 2

[0043] Compared with Example 1, except that the cofactor in step 4 is 0.05-0.6mM 5-ALA, the others are the same.

Embodiment 3

[0045] Compared with Example 1 except that the cofactor in step 4 is 0.01-0.4mM Fe 3+ Except for the difference, everything else is the same.

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Abstract

The invention relates to cytochrome P450 enzymes, in particular to a method for improving the in-vitro expression of a cytochrome P450 enzyme family. The method for improving the in-vitro expression of the cytochrome P450 enzyme family is characterized in that a cofactor in step D is any one of the following components: (1) Hemin with the concentration of between 0.5 and 6mu g / ml; (2) 5-ALA at the concentration of between 0.05 and 0.6mM; (3) Fe<3+> at the concentration of between 0.01 and 0.4mM; and (4) a mixture of the 5-ALA at the concentration of between 0.05 and 0.6mM and the Fe<3+> at the concentration of between 0.01 and 0.4mM. System optimization is performed on cytochrome P450 (CYP) protein, P450 oxidoreductase (POR) protein and coexpression conditions thereof for the first time, and the result shows that the expression levels of the proteins and the activities thereof are highest when the Fe<3+> at the concentration of 0.02mM and the 5-ALA at the concentration of 0.2mM are synchronously given to treat cells. When the multiplicity of infection (MOI) of CYP is 5pfu / cell and the MOI of POR is 2pfu / cell, the coexpression efficiency of the CYP and the POR is highest.

Description

[0001] technical field [0002] The invention relates to cytochrome P450 enzymes, in particular to a method for improving the expression of cytochrome P450 enzyme family in vitro. Background technique [0003] Cytochrome P450 (CYP450) enzyme is the terminal oxidase of the mixed-function oxidase system on the endoplasmic reticulum membrane. Intermediate or final products with strong electrical properties can cause a series of toxic effects. Existing research on CYP450 enzymes metabolically activates exogenous compounds mainly includes in vivo and in vitro metabolism. In vivo metabolism studies are represented by animal models, but there are great differences between different species of animals and humans, especially in terms of metabolism, the types of enzymes involved in compound metabolism, the degree of contribution of enzymes, reaction types, and metabolites. It may not be the same, and the extrapolation of animal experiment results to human body has high error and lo...

Claims

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Application Information

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IPC IPC(8): C12Q1/26C12N15/09C12N15/53
Inventor 王守林陆慧媛
Owner NANJING MEDICAL UNIV
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