CN101928749A

CN101928749A - Method for detecting S-adenosylmethionine (AdoMet)-dependent methyltransferase

Info

Publication number: CN101928749A
Application number: CN2010101978085A
Authority: CN
Inventors: 谷劲松; 叶春江
Original assignee: University of Jinan
Current assignee: Jinan University; University of Jinan
Priority date: 2010-06-11
Filing date: 2010-06-11
Publication date: 2010-12-29

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

The detection method of the methyltransgerase that a kind of S-adenosylmethionine (AdoMet) relies on

Technical field

The invention belongs to the biochemical analysis field, relate to the detection method of the methyltransgerase of a kind of S-adenosylmethionine (AdoMet) dependence.More specifically, the present invention relates to obtain S-adenosyl-L-homocysteine nucleosidase (MTAN by gene engineering method, EC 3.2.2.9) and reorganization S-Ribosylhomocysteinase (LuxS, EC3.2.1.148) two kinds of recombinases, and set up the methyltransgerase analysis that the enzyme coupling technology is used for S-adenosylmethionine (AdoMet) dependence.

Background technology

No matter the methyltransgerase that S-adenosylmethionine (AdoMet) relies on still be all extensively existence of eukaryote prokaryotic organism, this enzyme family member is above 130 kinds, substrate (target spot) scope of their effects has almost been included all bioactive moleculess in the cell, comprise nucleic acid, protein, polysaccharide, lipid and many small molecules substrates, bioprocesss such as its catalytic methyl modification reaction is synthetic to biomacromolecule, signal conduction, protein reparation, chromosomal expression regulation and control, gene silencing play vital regulating effect.

At present, it is radioactive that methyl transferase activity detects many employings ¹⁴C or ³H mark AdoMet, radiolabeled product is observed by the radioisotope scan imager in Methyl transporters reaction back.This method needs loaded down with trivial details follow-up chromatographic separation substrate and product, waste time and energy, and poor repeatability, can only accomplish semi-quantitative analysis under most of situation, the more important thing is that the product S-adenosine-L homocysteine (AdoHcy) behind the AdoMet transmethylase has significant inhibitory effect to the methyltransgerase that AdoMet relies on, thereby influenced the accurate metering of enzyme reaction parameter. ¹⁴C or ³The price of H mark AdoMet costliness, the expensive processing costs of radwaste also is the shortcoming that this method is difficult to overcome.

Summary of the invention

The enzyme coupling analysis and detection technology that the purpose of this invention is to provide the methyltransgerase that a kind of accurately stable, safe and reliable AdoMet relies on.

For achieving the above object, the present invention is by the following technical solutions:

Set up enzyme coupling analysis and detection technology based on reorganization S-adenosyl-L-homocysteine nucleosidase (MTAN, EC 3.2.2.9) and reorganization S-Ribosylhomocysteinase (LuxS, EC 3.2.1.148).AdoMet generates S-adenosyl-L-homocysteine (AdoHcy) by methyltransgerase after by transmethylase, product A doHcy is by MTAN (reorganization S-adenosyl-L-homocysteine nucleosidase) and two excessive recombinase catalysis of LuxS (reorganization S-Ribosylhomocysteinase), after the two step scission reactions, be converted into homocysteine (homocycteine) fully, the latter uses Ellman reagent 5,5 '-Lian sulphur-2,2 '-two nitrobenzoic acids (DTNB) are quantitative.As shown in Figure 1:

LuxS and MTAN be and two kinds of genetically engineered recombinases of methyltransgerase link coupled, and these two kinds of zymoprotein sequence of N ends or C-terminal are connected with histidine-tagged (His-tagged), and we are by nickel ion (Ni ²⁺) absorption of agarose affinity chromatography post, the imidazoles wash-out obtains two recombinases of methyltransgerase enzyme linked reaction.

The present invention has not only overcome when using radio-labeling AdoMet to analyze methyltransgerase, time-consuming, the sxemiquantitative that has, poor repeatability and reactant need loaded down with trivial details shortcomings such as later separation step, and eliminated the feedback inhibition of Methyl transporters product A doHcy, methylation reaction can be carried out fully.We can adopt this inventive method that the enzymatic reaction process of Methyl transporters is carried out detailed enzymatic reaction kinetics research, and inquire into its optimum reaction condition and influence factor, and the present invention has guaranteed that we are to methyl transferase activity quantitative analysis accurately and effectively.

Description of drawings

Fig. 1 shown by with two kinds of genetically engineered recombinases of methyltransgerase link coupled AdoHcy nucleosidase (MTAN, EC 3.2.2.9) and reorganization S-Ribosylhomocysteinase (LuxS, the EC 3.2.1.148) reaction mechanism of coming the universal product S-adenosyl-L-homocysteine (AdoHcy) of analyzing and testing methyltransgerase.

Fig. 2 A is a gel electrophoresis figure, purity and productive rate by sodium lauryl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) check enzyme coupling analyzing and testing two recombinase LuxS of system, having 6 histidine-tagged LuxS molecular weight of albumen is 19.0kDa, because 84 cysteine residues can be oxidized, so be rendered as two bands.

Fig. 2 B is a gel electrophoresis figure, and by purity and the productive rate of sodium lauryl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) check enzyme coupling analyzing and testing two recombinase MTAN of system, having 6 histidine-tagged MTAN molecular weight of albumen is 25.3kDa.

Fig. 3 illustrates the stoichiometry that the enzyme linked reaction transforms the 5-sulfo--2-nitrobenzoic acid (TNB) of S-adenosyl-L-homocysteine (AdoHcy) generation.Because the Guanidinium hydrochloride termination reaction of 4 times of reaction volumes of enzyme linked reaction ultimate demand, so the volumetric molar concentration of the 5-sulfo--2-nitrobenzoic acid (TNB) that generates should be 20% of SAH volumetric molar concentration.

Embodiment:

Embodiment 1

The expression and purification of reorganization S-adenosyl-L-homocysteine nucleosidase (MTAN), its process may further comprise the steps:

I. express: adopting the primer that comprises NdeI and BamHI endonuclease digestion site is polymerase chain reaction (PCR) the product rMTAN-8 that template amplification lacks the 675bp of preceding 8 amino acid coding with intestinal bacteria XL1-Blue chromosomal DNA, the PCR product cloning is gone into pCRII (InVitrogen) plasmid vector, recombinant plasmid transformed competence colibacillus cell intestinal bacteria TOP10F '.Alkaline lysis is from positive colony (white, ammonia benzyl resistance) prepares recombinant plasmid in a small amount, agarose electrophoresis is separated behind NdeI/BamHI (10 unit of enzyme/37 ℃/12 hours) double digestion, the separation and purification endonuclease bamhi is cloned into tryptophane inducible expression carrier pAL781 (InVitrogen) from glue, transformed competence colibacillus cell intestinal bacteria GI728, transformant is inoculated in Luria-Bertani (LB) nutrient solution that contains 100 μ g/mL penbritins 37 ℃, and the 225rpm shaking culture is to OD ₆₀₀Reach 0.5-0.6,0.1mM tryptophane abduction delivering MTAN (rMTAN-8).

Ii. purifying: centrifugal collection thalline, 1 * PBS damping fluid are given a baby a bath on the third day after its birth time, ultrasonic treatment centrifugal (30,000g) go precipitation, lysate is splined on Ni ²⁺The agarose affinity chromatography post, with containing 20mM potassiumphosphate, 0.1M NaCl, and pH 7.8 solution of 5mM or 10mM imidazoles wash 5-6 column volume, use 20mM potassiumphosphate, 0.1M NaCl at last, and 500mM imidazoles pH 7.8 elutriant wash-outs, SDS-PAGE determines productive rate and purity (seeing accompanying drawing 2B), and having 6 histidine-tagged MTAN molecular weight of albumen is 25.3kDa.

Embodiment 2

The expression and purification of reorganization S-Ribosylhomocysteinase (LuxS), its process may further comprise the steps:

I. express: with the subtilis genomic dna is template, employing comprises the primer PCR amplification LuxS gene in NdeI and NotI endonuclease digestion site, the PCR product cloning is gone into (Novagen) expression vector of pET29a (+), transformed competence colibacillus cell e. coli bl21 (DE3), recombinant plasmid pLuxSH ₆The coding C-terminal has 6 histidine-tagged products.Transformant is inoculated in the LB nutrient solution that contains 40 μ g/mL kantlex 37 ℃, and the 225rpm shaking culture is to OD ₆₀₀Reach 0.5-0.6,0.4mM sec.-propyl-b-D-thiogalactoside (IPTG) abduction delivering S-Ribosylhomocysteinase (LuxS).

Ii. purifying: centrifugal collection thalline, 1 * PBS damping fluid are given a baby a bath on the third day after its birth time, ultrasonic treatment centrifugal (30,000g) go precipitation, lysate is splined on Ni ²⁺The agarose affinity chromatography post, with containing 20mM potassiumphosphate, 0.1M NaCl, and pH 7.8 solution of 5mM or 10mM imidazoles wash 5-6 column volume, use 20mM potassiumphosphate, 0.1M NaCl at last, and 500mM imidazoles pH 7.8 elutriant wash-outs, SDS-PAGE determines productive rate and purity (seeing accompanying drawing 2A), having 6 histidine-tagged LuxS molecular weight of albumen is 19.0kDa, because 84 cysteine residues can be oxidized, so be rendered as two bands.

Embodiment 3

Based on the foundation of the enzyme coupling analysis and detection technology of the reorganization S-adenosyl-L-homocysteine nucleosidase (MTAN, EC 3.2.2.9) and the S-Ribosylhomocysteinase (LuxS, EC 3.2.1.148) of recombinating, its process may further comprise the steps:

The drafting of the typical curve of 5-sulfo--2-nitrobenzoic acid (TNB) that i.S-adenosine-L-homocysteine (AdoHcy) and colorimetric estimation generate: enzyme coupling analyzing and testing system includes 0,17,34,51,68,85,102,119 S-adenosyl-L-homocysteines such as different concns such as grade (AdoHcy), and 1.5 μ M S-adenosyl-L-homocysteine nucleosidase (MTAN), 10 μ M S-Ribosylhomocysteinases (LuxS), the 100mM Hepes damping fluid of pH 8.0,37 ℃ were reacted 30 minutes, with tetraploid long-pending 5,5 '-Lian sulphur-2,2 '-two nitrobenzoic acids (DTNB) (133 μ M)-Guanidinium hydrochloride (8M) solution termination reaction, 5-sulfo--2-nitrobenzoic acid (TNB) that the 412nm colorimetric estimation generates.Experimental result shows, in enzyme coupling analyzing and testing system, two recombinases that recombinant expressed purifying obtains all present good catalytic, and 5-sulfo--2-nitrobenzoic acid (TNB) that the enzyme linked reaction generates presents significant linear positive correlation (accompanying drawing 3) with S-adenosyl-L-homocysteine (AdoHcy) concentration.

Ii. enzyme coupling analysis and detection technology detects methyl transferase activity: enzyme coupling analyzing and testing system includes methyltransgerase to be measured, 430 μ M S-adenosylmethionines (AdoMet), 1.5 μ M S-adenosyl-L-homocysteine nucleosidase (MTAN), 10 μ M S-Ribosylhomocysteinases (LuxS), the 100mMHepes damping fluid of pH 8.0,37 ℃ were reacted 30 minutes, with tetraploid long-pending 5,5 '-Lian sulphur-2,2 '-two nitrobenzoic acids (DTNB) (133 μ M)-Guanidinium hydrochloride (8M) solution termination reaction, 5-sulfo--2-nitrobenzoic acid (TNB) that the 412nm colorimetric estimation generates calculates S-adenosyl-L-homocysteine (AdoHcy) amount of transmethylase generation according to the S-adenosyl-L-homocysteine (AdoHcy) and the typical curve of 5-sulfo--2-nitrobenzoic acid (TNB).

Claims

1. the enzyme coupling analysis and detection technology of the methyltransgerase that relies on of accurately stable, a safe and reliable S-adenosylmethionine (AdoMet), it is characterized in that: by adopting reorganization S-adenosyl-L-homocysteine nucleosidase (MTAN, EC 3.2.2.9) and the universal product S-adenosyl-L-homocysteine (AdoHcy) that generates behind reorganization S-Ribosylhomocysteinase (LuxS, EC 3.2.1.148) these two kinds of enzyme coupling analyzing and testing transmethylases analyze the methyl transferase activity that S-adenosylmethionine (AdoMet) relies on.

2. according to the described enzyme coupling of claim 1 analysis and detection technology, it is characterized in that: the object that this technology is used is the methyltransgerase that S-adenosylmethionine (AdoMet) relies on.

Reorganization S-adenosyl-L-homocysteine nucleosidase 3. according to claim 1 (MTAN EC3.2.2.9), is characterized in that:

(1) N-terminal has 6 histidine-tagged recombinant expression proteins, and molecular weight is 25.3kDa;

(2) by nickel ion (Ni ²⁺) absorption of agarose affinity chromatography post, the imidazoles wash-out obtains the recombinase of methyltransgerase enzyme linked reaction;

(3) its coding gene sequence is polymerase chain reaction (PCR) the product rMTAN-8 of 675bp;

(4) its coding gene sequence is gone into tryptophane inducible expression carrier pAL781 (InVitrogen) by subclone, transformed competence colibacillus cell intestinal bacteria GI728, transformant are inoculated in shaking culture tryptophane abduction delivering acquisition in Luria-Bertani (LB) nutrient solution that contains penbritin.

4. the PCR product rMTAN-8 of 675bp according to claim 3 is that the primer that comprises NdeI and BamHI endonuclease digestion site by employing is that the template pcr amplification comes with intestinal bacteria XL1-Blue chromosomal DNA.

5. reorganization S-Ribosylhomocysteinase according to claim 1 (LuxS, EC 3.2.1.148) is characterized in that:

(1) C-terminal has 6 histidine-tagged recombinant expression proteins, and molecular weight is 19.0kDa;

(3) its coding gene sequence is polymerase chain reaction (PCR) the product LuxS of 504bp;

(4) its coding gene sequence is cloned into (Novagen) expression vector of pET29a (+), and transformed competence colibacillus cell e. coli bl21 (DE3), transformant are inoculated in the LB nutrient solution that contains kantlex, shaking culture, and the IPTG abduction delivering obtains.

6. the polymerase chain reaction of 504bp according to claim 5 (PCR) product LuxS is to be template with the subtilis genomic dna, adopts the primer PCR that comprises NdeI and NotI endonuclease digestion site to increase.

7. enzyme coupling analysis and detection technology according to claim 2 is characterized in that:

(1) during concrete operations except comprising methyltransgerase to be measured, also to add following component: S-adenosylmethionine (AdoMet), S-adenosyl-L-homocysteine nucleosidase (MTAN), S-Ribosylhomocysteinase (LuxS), Hepes damping fluid;

(2) reaction conditions is 37 ℃ of reactions 30 minutes;

(3) with total reaction liquid tetraploid long-pending 5,5 '-Lian sulphur-2,2 '-two nitrobenzoic acids (DTNB)-guanidine hydrochloride solution termination reaction, 5-sulfo--2-nitrobenzoic acid (TNB) that colorimetric estimation generates.