Check patentability & draft patents in minutes with Patsnap Eureka AI!

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

An in vitro expression, cytochrome technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, plant genetic improvement, etc., can solve the problems of low yield and difficult to meet research needs, and achieve considerable economic benefits and a good society. effect of benefit

Inactive Publication Date: 2013-01-02
NANJING MEDICAL UNIV
View PDF1 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • 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.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to a cytochrome P450 enzyme, and especially relates to a method for improving in-vitro expression of a cytochrome P450 enzyme family. The method for improving in-vitro expression of the cytochrome P450 enzyme family is characterized in that an accessory factor in step D any one of the followings: (1) 0.5-6 microgram / ml Hemin; (2) 0.05-0.6 mM 5-ALA; (3) 0.01-0.4 mM Fe3+; and (4) a mixture of 0.05-0.6 mM 5-ALA and 0.01-0.4 mM Fe3+. CYC protein, POR protein and their coexpression conditions are systemically optimized for the first time; and the results show that when cells are treated by 0.03 mM Fe3+ and 0.2 Mm 5-ALA, the expression levels of the proteins and their activities are the highest. When the MOI of CYP is 5 pfu / cell, and the MOI of POR is 2 pfu / cell, the coexpression efficiency of the CYP and POR is the 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N15/866C12N7/01C07K14/47
Inventor 王守林陆慧媛
Owner NANJING MEDICAL UNIV
Features
  • R&D
  • Intellectual Property
  • Life Sciences
  • Materials
  • Tech Scout
Why Patsnap Eureka
  • Unparalleled Data Quality
  • Higher Quality Content
  • 60% Fewer Hallucinations
Social media
Patsnap Eureka Blog
Learn More

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com