Recombinant escherichia coli for expression of adenomethionine synthetase

A technology of Escherichia coli and adenosylmethionine, which is applied in the biological field and can solve problems such as difficult separation

Active Publication Date: 2009-03-25
BEIJING KAWIN TECH SHARE HLDG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The chemical synthesis method uses S-adenosythomocysteine ​​(S-adenosythomocysteine) and methyl donor (CH3I) to synthesize, adenosylmethionine has two configurations (+) and (-), only (-) configuration It has biological activity, but the synthetic product contains a large amount of (+) adenymethionine, and it is difficult to separate

Method used

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  • Recombinant escherichia coli for expression of adenomethionine synthetase
  • Recombinant escherichia coli for expression of adenomethionine synthetase
  • Recombinant escherichia coli for expression of adenomethionine synthetase

Examples

Experimental program
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Embodiment 1

[0030] Example 1 Construction of recombinant Escherichia coli expressing adenosylmethionine synthetase

[0031] 1. Construction of recombinant expression vector pMETK:

[0032] The metK gene has been disclosed in literature (J.Biol.Chem.259(23), 14505-14507(1984))

[0033] (1) Inoculate E.coli DH5α onto LB solid medium, culture at 37°C for 18 hours, then use a sterilized toothpick to pick a single colony and inoculate it into 50ml LB liquid medium, cultivate overnight at 37°C and 180rpm shaker, Then the genomic DNA was extracted with a DNA extraction kit.

[0034] (2) Entrust Shanghai Sangon Bioengineering Co., Ltd. to synthesize the following primers:

[0035] Forward primer sequence:

[0036] 5'-AA CTGCAG AGTCGTGGTAGGATCCGCTACCACA-3',

[0037] Reverse primer sequence:

[0038] 5'-G GAATTC ATCTGCAATAAACGGCAGTGCGC-3';

[0039] (3) Take 1 μg of DNA template, add 2 μmol / L of primers in step (2), 5 IU of Taq DNA polymerase, 200 μmol / L of dNTPs, 20 μL of 1×PCR buffer, mi...

Embodiment 2

[0144] Expression amount and activity determination of the recombinant Escherichia coli adenosylmethionine synthetase constructed in Example 2

[0145] experiment

[0146] (1) Determination of the expression level of adenosylmethionine synthetase

[0147] Pick the single bacterium colony of the recombinant Escherichia coli (Escherichia coli) CGMCCNo.2299 that embodiment 1 obtains, inoculate into 10ml liquid LB culture medium (containing the oxytetracycline of 25 μ g / ml), shake table culture 6 hours, take a sample with SDS -PAGE polyacrylamide electrophoresis to determine the expression level of adenosylmethionine synthase, the electrophoresis results are as attached image 3 As shown, the expression level of SAM synthetase accounted for 31.36% of the soluble protein of the bacteria.

[0148] (2) Determination of the activity of adenosylmethionine synthase

[0149] Preparation of reaction solution: from 75mmol Tris-HCl (PH8.0), 250mmol KCl, 9mmol MgCl 2 , 60μmol methionine,...

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Abstract

The invention provides a recomposed large intestine Escherichia coli expressing adenosine methionine synthetase with CGMCC No. 2299 and the composition method of the recomposed large intestine Escherichia coli. The invention further provides a recomposed expression carrier pMETK of adenosine methionine synthetase. In the recomposed large intestine Escherichia coli expressing adenosine methionine synthetase with CGMCC No. 2299, the expression quantity of the adenosine methionine synthetase accounts for 31.36 percent of soluble albumen of the strain, the activity of the adenosine methionine synthetase expressed is as high as 7.38U / ml, far higher than that of wild type strains.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant Escherichia coli expressing adenosylmethionine synthetase and its application. Background technique [0002] China is a country with a large population, but also a "big hepatitis country". According to the national viral hepatitis epidemiological survey in 1992, about 120 million people in the country carry hepatitis B virus, and the infection rate of hepatitis C in the general population is 3.1%. There are currently 20 million chronic hepatitis B patients in my country, some of whom may transform into liver cirrhosis or even liver cancer. [0003] Adenosylmethionine (SAM) is an extremely important intermediate metabolite in the human body. It can generate the key antioxidant and detoxification substance in the body-cysteine ​​through the transsulfurization pathway, and regenerate another key antioxidant and detoxification substance-glutathione to relieve the oxidativ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/52C12N15/70C12N9/00C12P13/04C12R1/19
Inventor 张春丽周德胜吉春谭华征
Owner BEIJING KAWIN TECH SHARE HLDG
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