Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Method for detecting S-adenosylmethionine (AdoMet)-dependent methyltransferase

An adenosylmethionine, methyltransferase technology, applied in the field of biochemical analysis, can solve the problems of AdoMet's expensive price, time-consuming and laborious, affecting the accurate measurement of enzyme reaction parameters, achieve accurate and effective quantitative analysis, and eliminate feedback inhibition. Effect

Inactive Publication Date: 2010-12-29
UNIV OF JINAN
View PDF2 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method requires cumbersome follow-up chromatographic separation of substrates and products, which is time-consuming and laborious, and has poor repeatability. In most cases, only semi-quantitative analysis can be achieved. More importantly, the product S-adenosine-L after AdoMet transmethylation is high Cysteine ​​(AdoHcy) has a significant inhibitory effect on AdoMet-dependent methyltransferases, thereby affecting the accurate measurement of enzyme reaction parameters
14 C or 3 H marks the high price of AdoMet, and the high cost of radioactive waste treatment is also a shortcoming of this method that is difficult to overcome

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 detecting S-adenosylmethionine (AdoMet)-dependent methyltransferase
  • Method for detecting S-adenosylmethionine (AdoMet)-dependent methyltransferase
  • Method for detecting S-adenosylmethionine (AdoMet)-dependent methyltransferase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] The expression and purification of recombinant S-adenosine-L-homocysteine ​​nuclease (MTAN) includes the following steps:

[0015] i. Expression: using primers containing NdeI and BamHI endonuclease cleavage sites and using E. coli XL1-Blue chromosomal DNA as a template to amplify the 675bp polymerase chain reaction (PCR) product rMTAN- which lacks the first 8 amino acid codes 8. The PCR product was cloned into the pCRII (InVitrogen) plasmid vector, and the recombinant plasmid was transformed into competent cells of Escherichia coli TOP10F'. Alkaline lysis method prepares a small amount of recombinant plasmids from positive clones (white, ampicillin resistant), NdeI / BamHI (10 enzyme units / 37°C / 12 hours) double enzyme digestion, agarose electrophoresis, separation and purification of the digested fragments from the gel The tryptophan inducing expression vector pAL781 (InVitrogen) was cloned into the competent cell E. coli GI728, and the transformant was inoculated in Luria-...

Embodiment 2

[0018] The expression and purification of recombinant S-ribosyl homocysteinase (LuxS) includes the following steps:

[0019] i. Expression: Using Bacillus subtilis genomic DNA as a template, the LuxS gene was amplified by PCR with primers containing NdeI and NotI endonuclease cleavage sites, and the PCR product was cloned into the pET29a(+) (Novagen) expression vector to transform competent Cell E. coli BL21(DE3), recombinant plasmid pLuxSH 6 It encodes a product with 6 histidine tags at the C-terminus. The transformant was inoculated into LB broth containing 40μg / mL kanamycin and cultured with shaking at 225rpm to OD at 37°C 600 To reach 0.5-0.6, 0.4mM isopropyl-b-D-thiogalactoside (IPTG) induces the expression of S-ribosyl homocysteinase (LuxS).

[0020] ii. Purification: collect bacteria by centrifugation, wash three times with 1×PBS buffer, ultrasonic lysis centrifugation (30,000g) to remove precipitation, and load the lysate on Ni 2+ Agarose affinity chromatography column, was...

Embodiment 3

[0022] Enzyme coupling analysis based on recombinant S-adenosyl-L-homocysteine ​​nuclease (MTAN, EC 3.2.2.9) and recombinant S-ribosyl homocysteine ​​(LuxS, EC 3.2.1.148) The establishment of detection technology includes the following steps:

[0023] The standard curve of iS-adenosine-L-homocysteine ​​(AdoHcy) and 5-thio-2-nitrobenzoic acid (TNB) generated by colorimetric determination: Enzyme coupling analysis detection system contains 0 , 17, 34, 51, 68, 85, 102, 119 and other different concentrations of S-adenosine-L-homocysteine ​​(AdoHcy), and 1.5μM S-adenosine-L-homocysteine ​​nucleoside Enzyme (MTAN), 10μM S-ribosyl homocysteinase (LuxS), 100mM Hepes buffer pH 8.0, reacted at 37°C for 30 minutes, with four times the volume of 5,5'-dithio-2,2 '-Dinitrobenzoic acid (DTNB) (133μM)-guanidine hydrochloride (8M) solution was used to terminate the reaction, and the generated 5-thio-2-nitrobenzoic acid (TNB) was measured colorimetrically at 412nm. The experimental results show ...

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

PropertyMeasurementUnit
Protein molecular weightaaaaaaaaaa
Molecular weightaaaaaaaaaa
Login to View More

Abstract

The invention discloses a method for detecting S-adenosylmethionine (AdoMet)-dependent methyltransferase, which is established based on the enzyme coupling analysis detection technology of recombinant S-adenosyl-L-homocysteine nucleosidase and recombinant S-ribosyl homocysteine. The invention is characterized by providing an accurate, stable, safe and reliable enzyme coupling analysis detection technology for AdoMet-dependent methyltransferase, which not only overcomes the shortcomings of time consumption, semi-quantitativeness, weak repeatability, complicated subsequent reactant separation steps and the like caused by using radioactive marker AdoMet to analyze the methyltransferase, but also eliminates feedback inhibition of the methyl transfer product S-adenosyl-L-homocysteine (AdoHcy) so that the methylation reaction can be performed completely, thus the enzyme coupling analysis detection system can certainly be used for performing detailed study on enzyme reaction dynamics for the catalytic reaction process of the methyltransferase.

Description

Technical field [0001] The invention belongs to the field of biochemical analysis and relates to a detection method of S-adenosylmethionine (AdoMet)-dependent methyltransferase. More specifically, the present invention relates to obtaining S-adenosyl-L-homocysteine ​​nuclease (MTAN, EC 3.2.2.9) and recombinant S-ribosyl homocysteinase (LuxS, EC3.2.1.148) two recombinases, and established enzyme coupling technology for S-adenosylmethionine (AdoMet)-dependent methyltransferase analysis. Background technique [0002] S-adenosylmethionine (AdoMet)-dependent methyltransferases are widespread in both prokaryotes and eukaryotes. The enzyme family has more than 130 members, and the range of substrates (targets) they act on almost covers It covers all biologically active molecules in cells, including nucleic acids, proteins, polysaccharides, lipids and many small molecule substrates. The methyl modification reactions catalyzed by them can affect the synthesis of biological macromolecules...

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): C12Q1/48C12Q1/34C12N9/24
Inventor 谷劲松叶春江
Owner UNIV OF JINAN
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com