S-adenosylmethionine synthetase preparation and preparation method and application thereof

An adenosylmethionine and enzyme preparation technology, which is applied in the field of S-adenosylmethionine synthase enzyme preparations and can solve problems such as interference and poor stability

Active Publication Date: 2016-06-01
BEIJING STRONG BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the defect of cystathionine cycle enzyme method is that it is easily interfered by human endogenous cystathionine
The cycle enzymatic method of methyltransferase and hydrolase is not interfered by human endogenous cystathionine, but there are still disadvantages such as poor stability

Method used

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  • S-adenosylmethionine synthetase preparation and preparation method and application thereof
  • S-adenosylmethionine synthetase preparation and preparation method and application thereof
  • S-adenosylmethionine synthetase preparation and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1: Provide target sequence

[0068] According to the nucleotide sequence of S-adenosylmethionine synthetase published on the NCBI website (Genbank accession number is CP007391.1) is shown as SEQ ID NO:2.

[0069] Design primers, use the Escherichia coli genome as a template, and amplify the gene fragment of S-adenosylmethionine synthetase by PCR;

[0070] The PCR reaction system is as follows:

[0071] wxya 2 O35μl; 10×Buffer5μl; dNTP2μl; template DNA 2μl; forward primer 2.5μl; reverse primer 2.5μl; Taq enzyme 1μl;

[0072] The PCR reaction conditions are as follows:

[0073] 94°C for 3.5min; 94°C for 40s, 55°C for 40s, 72°C for 70s, a total of 35 cycles; 72°C for 10min; 4°C for maintenance;

[0074] Primer sequence:

[0075] Forward: GGGAATTCCATATGATGGCAAAACACCTTTTTAC (SEQ ID No. 3);

[0076] Reverse: ATCCGCTCGAGCTTCAGACCGGCAGCATCGC (SEQ ID No. 4).

[0077] Take the PCR product, and after 1% agarose gel electrophoresis, a product band can be seen at 1200...

Embodiment 2

[0079] Embodiment 2: Construction of expression vector

[0080] The PCR product recovered in Example 1 was subjected to double enzyme digestion with NdeI and XhoI enzymes (purchased from NewEngland Biolabs);

[0081] The vector plasmid pET41a (Novagen) was subjected to the same double enzyme digestion treatment;

[0082] The digested plasmid and gene were ligated overnight at 16°C;

[0083]Transform Escherichia coli DH5α the next day, and apply Kan (kanamycin, 50mg / L) resistant LB plates for screening;

[0084] The positive clones obtained by screening were extracted and sequenced after extraction of plasmids, and the plasmids with correct sequencing results were stored for later use.

Embodiment 3

[0085] Embodiment 3: transformation Escherichia coli BL21 (DE3)

[0086] The plasmid constructed in Example 2 was mixed with chemically competent cells BL21(DE3) (purchased from Invitrogen) and then kept in an ice bath for 20 minutes; heat-shocked at 42°C for 90 seconds; quickly added to SOC medium (purchased from Invitrogen), and incubated at 37°C for 1 Paint the panels after hours.

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Abstract

The invention relates to an S-adenosylmethionine synthetase preparation and a preparation method and application thereof.The preparation method includes the steps that S-adenosylmethionine synthetase genes are constructed into a prokaryotic expression vector through the gene engineering technology, and escherichia coli is converted to construct recombinant host cells; high-yield thalli are obtained through batch fermentation of supplementary materials; then through the affinity chromatography technology, S-adenosylmethionine synthetase is purified, wherein the chromatographic purity of obtained protein is 90% or above.The invention further relates to the application of S-adenosylmethionine synthetase to preparation of a homocysteine diagnostic reagent.

Description

technical field [0001] The present disclosure relates to the field of biochemistry; in particular, it relates to an S-adenosylmethionine synthetase enzyme preparation and its production method and application. Background technique [0002] S-adenosylmethionine synthetase (hereinafter referred to as MAT) (EC2.5.1.6) is a very important enzyme in organisms. in the presence of Mg 2+ and K + When present, it catalyzes the reaction of L-Met with ATP to generate S-adenosylmethionine (SAM). SAM is an important intermediate metabolite in organisms and participates in various biochemical reactions in vivo. S-adenosylmethionine synthetase is an important raw material enzyme. [0003] The existing methods for producing S-adenosylmethionine synthase mainly use yeast systems (see 1-4), including Pichia pastoris and Saccharomyces cerevisiae. The disadvantage of this method is mainly that the production process is more complicated than the prokaryotic expression system, the yield is l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12Q1/48C12Q1/32C12Q1/34
CPCC12N9/1085C12Q1/32C12Q1/34C12Q1/48C12Y205/01006
Inventor 龚俊高长文高秋峰刘希
Owner BEIJING STRONG BIOTECH INC
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