CN106119217B - A kind of recombination buckwheat glutaredoxin and its preparation method and application - Google Patents
A kind of recombination buckwheat glutaredoxin and its preparation method and application Download PDFInfo
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Abstract
The present invention provides a kind of recombination buckwheat glutaredoxins and its preparation method and application.Preparation method: will recombination buckwheat glutaredoxin (rbGrx) it is gene constructed on pGEX-6p-1 carrier, be transformed into e. coli bl21 (DE3), inducing expression is connected with the fusion protein of GST；It is further purified after digestion using anion-exchange column and gel column, obtains the recombination rbGrx albumen of 98% or more purity.And then in pH 4.5,22%PEG3350 (W/V) and 0.1M CH3Under the conditions of the crystallization of COONa, culture obtains rbGrx albumen and glutathione (GSH) compound crystal, and its structure is measured using the method for X-ray single crystal diffraction, important amino acid residue relevant to catalytic activity can be told, clearly so as to purposefully regulate and control the vigor of its stability and enzyme.The present invention, which recombinates buckwheat glutaredoxin, has high thioltransferase activity, specific activity of enzyme 300U/mg.It can be applied in catalysis sulfydryl transfer reaction, crystal and its structure health care and in terms of also there is practical value.
Description
Technical field
The present invention relates to biotechnologys and Medicines and Health Product field, and in particular to glutaredoxin more specifically belongs to
A kind of recombination buckwheat glutaredoxin (recombinant buckwheat glutaredoxin, referred to as rbGrx) and its system
Preparation Method and application.
Background technique
Some protein can be in the cysteine of cysteine residues active site and corresponding disulphide in organism
Between quickly carry out reversible sulfydryl and disulfide bond reaction, to adjust the redox potential that cell includes the albumen of sulfydryl,
The reversible reduction reaction of disulfide bond is mainly the sulfhydryl oxidase of active site containing double sulfydryl functional areas (C-X-X-C) by a series of
Reductase, which mediates, to be completed.The system for being responsible for sulfhydryl oxidase reduction in organism mainly has thioredoxin system (Thioredoxin
) and glutaredoxin system (Glutaredoxin system) system.Grx and glutathione (GSH), glutathione reduction
Enzyme (glutathione reductase, GR) and NAD (P) H collectively form Grx system, are modified and are adjusted by reversible redox
The glutathione of ganglion cell, the formation and reduction for removing glutathione and disulfide bond, to maintain cell Redox stable state.
Grx can be divided into single sulfydryl (C-X-X-S) and double sulfydryl (C-X-X-C) forms by the quantity of cysteine contained by active site.Grx
Exercise multiple biological function in the cell, including internal Oxidative Stress, adjust transcription factor, reduction hydroascorbic acid,
Inhibit Apoptosis, participate in the thio processes such as thank.Glutaredoxin is sent out in the plants such as rice, spinach, arabidopsis, poplar
It is existing, but there is not been reported for the identification of buckwheat glutaredoxin (rbGrx) gene and the expression and purification of albumen and functional analysis.
Summary of the invention
The purpose of the present invention is to provide a kind of recombination buckwheat glutaredoxins and preparation method thereof and the albumen to urge
Change the application in sulfydryl transfer reaction.
Another object of the present invention is to provide a kind of recombination buckwheat glutaredoxin crystal and its method for crystallising, Yi Jichong
The purposes of group buckwheat glutaredoxin crystal and its structure.
To achieve the above object the invention provides the following technical scheme:
A kind of encoding gene recombinating buckwheat glutaredoxin rbGrx, nucleotides sequence are classified as SEQ ID NO:1.The base
Because being obtained by analysis buckwheat complete genome sequence screening.It is compared with the sequence of the Grx albumen from not kindred plant
It was found that the Grx sequence homology of recombination buckwheat glutaredoxin rbGrx and other plant source, recombination buckwheat paddy oxygen also egg
White rbGrx is glutaredoxin family in plant, and belongs to the first albuminoid in family classification.
A kind of recombination buckwheat glutaredoxin encoded using gene as described above, amino acid sequence are SEQ ID
NO:2.It is made of 124 amino acid, and relative molecular weight is 13500 dalton.It goes back while capable of restoring protein disulfide bond
Can reduced glutathione albumen.
A kind of preparation method recombinating buckwheat glutaredoxin, includes the following steps:
(1) encoding gene of recombination buckwheat glutaredoxin rbGrx as described above is building up to pGEX-6p-1 carrier
On, it is transformed into e. coli bl21 (DE3), inducing expression is connected with the fusion protein of GST；
(2) it by the affine column separating purification GST-rbGrx albumen of Glutathione-sepharose, uses
ProScission protease digestion is incorporated in the gst fusion protein on affinity column, then also by the recombination buckwheat paddy oxygen after digestion
Albumen rbGrx is further purified using anion-exchange column and gel column, obtains recombination buckwheat glutaredoxin rbGrx.
The recombination buckwheat glutaredoxin rbGrx has very high thioltransferase activity, specific activity of enzyme 300U/
mg.Optimal pH is 8.5, and optimum temperature is 45 DEG C.The albumen can be applied in catalysis sulfydryl transfer reaction, in health care and food
Product exploitation etc. also has practical value.
A kind of method for crystallising recombinating buckwheat glutaredoxin crystal, step include: that will recombinate buckwheat glutaredoxin
RbGrx is concentrated into 6-7mg/ml, obtains crystal with sessile drop method under 18-25 degrees Celsius；Crystallize the condition of solution are as follows: 18%-
25%PEG3350 (W/V), pH 4.5-pH 5.0,0.1M CH3COONa。
The recombination buckwheat glutaredoxin crystal, for recombination buckwheat glutaredoxin rbGrx as described above and paddy
The compound crystal of the sweet peptide GSH of Guang；Space group with P2 (1) 2 (1) 2, cell parameter is about are as follows: α=β=γ=90 °；
The structure of the rbGrx crystal, the whole stereochemical structure that elliposoidal is similar in one are surrounded by 6 α spirals
It is formed around 4 β-pleated sheets；β 1,2,4 is antiparallel with 3；Wherein α spiral 1, i.e. amino acid containing Glu12-Ser28 section, β folding
Folded 1, i.e. the section of amino acid containing Leu31-Ser35, α spiral 2, i.e. amino acid containing Gly40-Leu52 section, β-pleated sheet 2 contains
Gln57-Glu60 amino acid section, α spiral 3, i.e. amino acid containing Leu61-Gln63 section, α spiral 4 contain Gly67-Thr78
Amino acid section, β-pleated sheet 3, i.e. amino acid containing Asn85-Ile88 section, β-pleated sheet 4, i.e. amino acid containing Lys91-Gly94 section,
α spiral 5, i.e. amino acid containing Cys96-Asp105 section, α spiral 6, i.e. amino acid containing Lys107-Cys115 section.
Based on the crystal structure of recombination buckwheat glutaredoxin rbGrx and GSH complex, it can clearly tell and live with catalysis
The relevant important amino acid full region of property, including K36, C39, Q71, T82VP84And G95CD97.So as to purposefully regulate and control it
The vigor of stability and enzyme, such as its activity for losing thioltransferase after mutation C39A.Meanwhile structure can be designed for drug and
Food development provides theoretical direction.
Detailed description of the invention
Fig. 1 is the sequence alignment figure of the Grx albumen from not kindred plant
Fig. 2 recombinates peak figure of the buckwheat glutaredoxin rbGrx Jing Guo gel column purification, and rbGrx is with list for gel separation display
Body form exists, and homogeneity is good.
Fig. 3 recombinates the enzyme activity determination figure of buckwheat glutaredoxin rbGrx, in figure: (A) rbGrx enzyme activity determination figure, wherein
Curve containing ball is negative control, and curve containing square is rbGrx enzyme activity curve；(B) Cys39 sports the obvious drop of activity after Ala
It is low；(C) influence of the temperature to enzyme activity；(D) influence of the pH to enzyme activity.
The crystal structure figure of Fig. 4 recombination buckwheat glutaredoxin rbGrx
The catalytic active center figure of Fig. 5 recombination buckwheat glutaredoxin rbGrx
Specific embodiment
The screening and analysis of the recombination buckwheat encoding glutaredoxin proteins gene of embodiment 1
The encoding gene of buckwheat glutaredoxin rbGrx is recombinated, nucleotides sequence is classified as SEQ ID NO:1.The gene is
It is obtained by analysis buckwheat complete genome sequence screening.Recombinate the buckwheat glutaredoxin rbGrx and Grx from not kindred plant
The sequence alignment (see Fig. 1) of albumen, including 12 kinds of Grx sequences of nine kinds of different plants.Wherein PoGRXC1 is used from beauty
State National Institutes database 2E7P_A, PoGRXS14 come from U.S. National Institutes database Accession:2LKU_A,
PoGRXS12 comes from U.S. National Institutes database Accession:3FZ9_A, and AtGRXS16 comes from U.S. National Institutes number
U.S. National Institutes database Accession:3IPZ_A, AtGRXC5 are come from according to library Accession:2LWF_A, AtGRXcp
From U.S. National Institutes database Accession:3RHB_A, OrGRX (Oryza sativa) from US National health
Institute database Accession:CAA54397, CuGRX (Cucumis sativus) come from U.S. National Institutes database
Accession:NP_001274139, IpGRX (Ipomoea batatas) come from U.S. National Institutes database
Accession:ABP48736, ThGRX (Theobroma cacao) come from U.S. National Institutes database Accession:
XP_007036260, RiGRX (Ricinus communis) come from U.S. National Institutes database Accession:
CAA89699。
By comparing analysis inventors have found that the recombination buckwheat glutaredoxin rbGrx and Grx in other plant source is orderly
Column homology, recombination buckwheat glutaredoxin rbGrx is glutaredoxin family in plant, and is belonged to first in family classification
Albuminoid.The amino acid deletions indicated in corresponding section are used ... in figure.The specific amino in specification and claims
Sour position is illustrated with the sequence of rbGrx.
Embodiment 2 recombinates expression and purifying of the encoding gene of buckwheat glutaredoxin in Escherichia coli.
The encoding gene for recombinating buckwheat glutaredoxin rbGrx pGEX-6p-1 is building up to by technique for gene engineering to carry
On body.The fusion protein expression plasmid of clone's building is transformed into BL21 (DE3), is first inoculated in the LB culture of 5mL based on 37
It DEG C is incubated overnight, after 12 hours, is transferred in the LB culture medium of 800mL and expands culture, culture to OD is in shaking flask at 37 DEG C
0.4-0.6 or so reduces cultivation temperature to 16 DEG C, the IPTG (isopropyl-β-D of final concentration of 0.5mM is then added after 4 hours
Thiogalactoside) inducing expression is carried out, bacterium is received in centrifugation after about 16-20 hours.With the HEPES buffer solution of 50mM (pH 7.8,
NaCl containing 200mM) suspension thalline, it is insoluble heavy by being centrifugated out using low-temperature ultrahigh-pressure cell crushing instrument lytic cell
It forms sediment, the supernatant that high speed centrifugation (about 13,000rmp) is obtained afterwards is by Glutathione-sepharose affinity column come preliminary point
From purifying GST-rbGrx albumen.Albumen containing GST label can be incorporated into Glutathione-Sepharose affinity column, make
Replaced with 50mM HEPEs (pH7.8), 500mM NaCl with 200mM NaCl buffer after cleaning foreign protein, is used
ProScission protease digestion is incorporated in the gst fusion protein on affinity column, and this process about 20 hours, then by digestion
The recombination buckwheat glutaredoxin rbGrx to get off using anion-exchange column and gel column be further purified out rbGrx albumen (see
Fig. 2), buckwheat glutaredoxin rbGrx purity is recombinated up to 98% or more.
The measurement of the recombination buckwheat glutaredoxin rbGrx enzyme activity of embodiment 3
The Grx vitality test of albumen: reaction system is 500 μ L, wherein including HEPEs (50mmol/L, pH 7.0), EDTA
(1mmol/L), GSH (1mmol/L), NADPH (0.25mmol/L), 1U GR (2 μm of ol/L).Reactant is mixed and is protected in advance
Final concentration 0.5mmol/L HED (bi-dihydroxy ethyl disulfide) starting reaction is added after warm 3min, monitors determinand 340nm
(εNADPH=6220M-1cm-1) at NADPH absorbance value variation.Grx vigor is defined as consuming 1 per minute under the conditions of 25 DEG C
An amount i.e. unit of activity for enzyme required for μm ol/L NADPH.The Rate activity of the rbGrx enzyme obtained by measurement is 300U/mg
± 15.0 (see Fig. 3).
The crystallization of 4 buckwheat glutaredoxin of embodiment and crystallographic structural analysis
The method for crystallising of buckwheat glutaredoxin is recombinated, step includes: to be concentrated into recombination buckwheat glutaredoxin rbGrx
7mg/ml obtains crystal with sessile drop method under 18 degrees Celsius；Crystallize the condition of solution are as follows: 22%PEG3350 (W/V), pH
4.5,0.1M CH3COONa。
Collection, processing and the structure elucidation of rbGrx diffraction data:
Diffraction data is collected using the X-ray source at Shanghai synchrotron radiation light source 18U line station, recombinates buckwheat glutaredoxin
RbGrx and GSH compound crystal diffraction resolution are up to 2.0 angstroms.Diffraction is handled using HKL2000 (Otwinowski 1997)
Data, discovery rbGrx and GSH complex proteins crystal have P2 (1) 2 (1) 2 space group, cell parameter are as follows:α=β=γ=90 °.
Structure elucidation is carried out using PHENIX program bag, carries out building and correcting for model by hand using software COOT.Into
After row simulated annealing, restricted atom offset position correction, energy minimize the amendment such as amendment, temperature factor, by having
The 2Fo-Fc differential electronic figure of sigma A weight factor adds solvent molecule.It can with geometry of the software PROCHECK to model
Confirmed by property.All images relevant to structure are shown and are made by software PyMOL.Finally tied
The R factor of structure is 16.8%, and the free R factor is 21.6% (see Fig. 4).
The crystal of the recombination buckwheat glutaredoxin rbGrx has the space group of P2 (1) 2 (1) 2, and cell parameter is about
Are as follows:α=β=γ=90 °.
Wherein the structure of the recombination buckwheat glutaredoxin rbGrx crystal is enclosed in 4 β-pleated sheets by 6 α spirals
Surrounding forms overall structure, and β 1,2,4 is antiparallel with 3.Wherein α spiral 1, i.e. amino acid containing Glu12-Ser28 section, β-pleated sheet
1, the i.e. section of amino acid containing Leu31-Ser35, α spiral 2, i.e. amino acid containing Gly40-Leu52 section, β-pleated sheet 2 contain
Gln57-Glu60 amino acid section, α spiral 3, i.e. amino acid containing Leu61-Gln63 section, α spiral 4 contain Gly67-Thr78
Amino acid section, β-pleated sheet 3, i.e. amino acid containing Asn85-Ile88 section, β-pleated sheet 4, i.e. amino acid containing Lys91-Gly94 section,
α spiral 5, i.e. amino acid containing Cys96-Asp105 section, α spiral 6, i.e. amino acid containing Lys107-Cys115 section.
Based on the crystal structure of recombination buckwheat glutaredoxin rbGrx and GSH complex, it can clearly tell and live with catalysis
The relevant important amino acid full region of property, including K36, C39, Q71, T82VP84And G95CD97(see Fig. 5).So as to there is mesh
The vigor for regulating and controlling its stability and enzyme, as being mutated its activity for losing thioltransferase after C39A.Meanwhile structure can be medicine
Object design and food development provide theoretical direction.
Claims (8)
1. a kind of encoding gene for recombinating buckwheat glutaredoxin rbGrx, nucleotides sequence are classified as SEQ ID NO:1.
2. the recombination buckwheat glutaredoxin of gene coding as described in claim 1, amino acid sequence is SEQ ID NO:
2。
3. a kind of preparation method for recombinating buckwheat glutaredoxin, includes the following steps:
(1) encoding gene of recombination buckwheat glutaredoxin rbGrx described in claim 1 is building up to pGEX-6p-1 carrier
On, it is transformed into e. coli bl21 (DE3), inducing expression is connected with the fusion protein of GST；
(2) by the affine column separating purification GST-rbGrx albumen of Glutathione-sepharose, ProScission egg is used
White enzyme digestion is incorporated in the gst fusion protein on affinity column, then uses the recombination buckwheat glutaredoxin rbGrx after digestion
Anion-exchange column is further purified with gel column, obtains recombination buckwheat glutaredoxin rbGrx.
4. a kind of recombination buckwheat glutaredoxin crystal, which is characterized in that the crystal is recombination as claimed in claim 2
The compound crystal of buckwheat glutaredoxin rbGrx and glutathione GSH；Space group with P2 (1) 2 (1) 2, cell parameter
Are as follows:α=β=γ=90 °；
The structure of the crystal is integrally in that the stereochemical structure for being similar to elliposoidal is enclosed in 4 β-pleated sheets by 6 α spirals
Surrounding is formed, and β 1,2,4 is antiparallel with 3, wherein α spiral 1, i.e., the section of amino acid containing Glu12-Ser28, β-pleated sheet 1 contain
Leu31-Ser35 amino acid section, α spiral 2, i.e. amino acid containing Gly40-Leu52 section, β-pleated sheet 2 contain Gln57-Glu60
Amino acid section, α spiral 3, i.e. amino acid containing Leu61-Gln63 section, α spiral 4, i.e. amino acid containing Gly67-Thr78 section,
β-pleated sheet 3, i.e. amino acid containing Asn85-Ile88 section, β-pleated sheet 4, i.e. amino acid containing Lys91-Gly94 section, α spiral 5 contain
Cys96-Asp105 amino acid section, α spiral 6, i.e. amino acid containing Lys107-Cys115 section.
5. application of the recombination buckwheat glutaredoxin in catalysis sulfydryl transfer reaction as claimed in claim 2.
6. recombination buckwheat glutaredoxin is preparing the application in Medicines and Health Product and food as claimed in claim 2.
7. application of the recombination buckwheat glutaredoxin crystal in structure elucidation as claimed in claim 4.
8. recombination buckwheat glutaredoxin crystal is designed in the activity and stability and drug of regulation enzyme as claimed in claim 4
With the application in food research and development.
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| CN101845443A (en) * | 2010-04-16 | 2010-09-29 | 浙江大学 | Tomato glutaredoxin gene SlRX1 as well as cloning method and application thereof |
| CN101965405A (en) * | 2008-01-25 | 2011-02-02 | 巴斯夫植物科学有限公司 | Plants having enhanced yield-related traits and a method for making the same |
| WO2013033571A1 (en) * | 2011-08-31 | 2013-03-07 | Kansas State University Research Foundation | Plants with enhanced tolerance to multiple abiotic stresses |
| CN104169424A (en) * | 2012-03-14 | 2014-11-26 | 纳幕尔杜邦公司 | Improving agronomic characteristics of plants through abph2 |
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| CN101845443A (en) * | 2010-04-16 | 2010-09-29 | 浙江大学 | Tomato glutaredoxin gene SlRX1 as well as cloning method and application thereof |
| WO2013033571A1 (en) * | 2011-08-31 | 2013-03-07 | Kansas State University Research Foundation | Plants with enhanced tolerance to multiple abiotic stresses |
| CN104169424A (en) * | 2012-03-14 | 2014-11-26 | 纳幕尔杜邦公司 | Improving agronomic characteristics of plants through abph2 |
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