CN109078617A - GST purified material, preparation method and product and its application - Google Patents
GST purified material, preparation method and product and its application Download PDFInfo
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Abstract
The present invention relates to field of biotechnology, specifically, providing a kind of GST purified material, preparation method and product and its application.GST purified material provided by the invention forms, wherein linking arm includes at least one of hexanediol diglycidyl ether or butanediol diglycidyl ether by solid phase carrier, linking arm and by the aglucon that linking arm is connect with solid phase carrier.The GST purified material is optimized by repetition test, is screened raw material meticulously and is obtained.Because linking arm has suitable molecular chain length and performance, is specifically bound so that GST is more advantageous to aglucon, improve the purification efficiency of material.The GST purified material is not only of fine quality but also inexpensive, can reuse repeatedly, compared with the similar product of current commercial type, with higher carrying capacity, absorption carrying capacity is big, and non-specific adsorption is few, purification effect is good, while applied widely, is suitble to large-scale device application.Preparation method is also simple controllable, can be widely applied.
Description
Technical field
The present invention relates to field of biotechnology, in particular to a kind of GST purified material, preparation method and product and
It is applied.
Background technique
Protein be constitute life material base and body vital movement the main undertaker, it is indispensable.With
Life science deepens continuously, and conducting a research to the structure and function of protein just becomes one of the hot spot of scientific research.
Obtaining a certain amount of high purity protein is the precondition to conduct a research to it, and certain albumen are in scientific research or production
Application be also required to its purity with higher.Obtaining the protein that people want using the method for genetic engineering increasingly becomes one
Plant convenient and effective method.Genetic engineering is that target gene is building up on expression vector, is then introduced into corresponding host
Inducing expression protein in cell.When carrying out protein recombinant expression, foreign protein high level expression often leads to be formed
Insoluble inclusion body will obtain active target protein and need by complicated change, renaturation process.Some albumen being capable of shape
At soluble expression product, but height is isolated from the mixture for being mixed with a large amount of thallus oneself proteins and other macromolecular substances
The target protein of purity is still the thing of part difficulty.
Many times, target protein and some large biological molecules or small peptide are subjected to amalgamation and expression for solving expression and pure
The problem of change is effective.These large biological molecules or small peptide are commonly referred to as fusion tag, wherein some labels can be with
Target protein is promoted to express in soluble form；Some labels can be specifically bound with other materials, to be subsequent point
Convenience is provided from purifying.
Glutathione sulfydryl transferase (glutathione S-transferase, GST) is common protein fusion expression
One of label.The expression of target protein not only can be improved in GST amalgamation and expression, but also for improving the solvable of target protein
Property expression also have certain help.Currently, GST label is in genetic engineering field wide application.
In conclusion producing one kind can seem particularly significant with the affinity chromatographic material of purified GST fusion protein.But
The GST affinity chromatographic material of commercial type or absorption carrying capacity are low at present, and non-specific adsorption is more, causes purification effect poor；
Expensive, production technology is complicated, is unfavorable for large-scale production.
In view of this, the present invention is specifically proposed.
Summary of the invention
The first object of the present invention is to provide a kind of GST purified material, to alleviate material or absorption in the prior art
Carrying capacity is low, and non-specific adsorption is more, causes purification effect poor；Expensive, production technology is complicated, is unfavorable for large-scale
The technical issues of production.
The second object of the present invention is to provide a kind of preparation method of GST purified material, be made in the prior art with alleviating
The technical issues of standby complex process, production cost is big or poor product quality.
The third object of the present invention is to provide a kind of GST purified product, and to alleviate, to lack a kind of GST in the prior art pure
Change that effect is good, simple and easy to operate while product at low cost.
The material or product that the fourth object of the present invention is to provide above-mentioned material or preparation method is prepared separation,
Application in purifying, analysis or identification GST albumen or gst fusion protein.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
A kind of GST purified material, including solid phase carrier, linking arm and the aglucon being connect by linking arm with solid phase carrier；
The linking arm includes hexanediol diglycidyl ether, neopentyl glycol diglycidyl glycerin ether or butanediol 2-glycidyl
At least one of ether.
Further, the solid phase carrier includes silica gel, controlled pore glass, sephadex, Ago-Gel or fibre
Tie up at least one of element, preferably at least one of Ago-Gel or sephadex, further preferably agarose
At least one of 4FF, agarose 6FF, agarose CL-4B or agarose CL-6B, still more preferably for
agarose 4FF。
Further, the aglucon is GSH albumen.
A kind of preparation method of GSH purified material, the preparation method include the steps that on ligand cou to linking arm,
Wherein, the linking arm is connect with solid phase carrier.
Further, the step of coupling includes: by aglucon, catalyst and solid phase carrier with linking arm in PBS
Incubation coupling is carried out in solution；
Preferably, the condition for being incubated for coupling includes: under the conditions of temperature is 45-55 DEG C, and 100-150rpm/min is incubated
Educate 12-20h；
Preferably, the concentration of the PBS solution is 10-30mM；
Preferably, the volume ratio of the solid phase carrier with linking arm and the PBS solution is 1:1-2；
Preferably, the concentration of the aglucon is 5.3-5.9g/L；
Preferably, the catalyst includes NaBH4Or KBH4, preferably NaBH4；
Preferably, the NaBH4Concentration be 1-1.4g/L.
Further, the step of linking arm is connect with solid phase carrier includes: by linking arm and solid phase carrier in alkalinity
Under the conditions of, incubation connection is carried out by catalyst；
Preferably, the volume ratio of the solid phase carrier and the linking arm is 1:0.4-0.8；
Preferably, the condition for being incubated for connection includes: under the conditions of 35-45 DEG C, and 100-150rpm/min is incubated for 5-
11h；
Preferably, the alkaline condition includes the organic solution containing NaOH or KOH, preferably organic molten containing NaOH
Liquid；
Preferably, the concentration of NaOH is 25-35g/L；
Preferably, the organic solution includes dimethyl sulfoxide or dimethylformamide, preferably dimethyl sulfoxide；
Preferably, the volume ratio of the solid phase carrier and the dimethyl sulfoxide is 1:1.2-1.6；
Preferably, the catalyst includes NaBH4Or KBH4, preferably NaBH4；
Preferably, NaBH4Concentration be 1-3g/L.
Further, the linking arm includes hexanediol diglycidyl ether, neopentyl glycol diglycidyl glycerin ether or butanediol
At least one of diglycidyl ether；
Preferably, the solid phase carrier includes silica gel, controlled pore glass, sephadex, Ago-Gel or fiber
At least one of element, preferably at least one of Ago-Gel or sephadex, further preferably agarose
At least one of 4FF, agarose 6FF, agarose CL-4B or agarose CL-6B, still more preferably for
agarose 4FF；
Preferably, the aglucon is GSH albumen.
Further, further include the steps that solid phase carrier deactivates after aglucon and linking arm coupling；
Described the step of deactivating includes: to be sealed the site not connecting with linking arm on solid phase carrier with ethanol amine
It closes；
Preferably, the closed condition includes: under the conditions of 35-40 DEG C, and 100-150rpm/min closes 12-20h；
Preferably, the solvent to deactivate is 3-7mM EDTA；
Preferably, the concentration of the ethanol amine is 0.5-1.5M；
Preferably, the volume ratio of the solid phase carrier and the ethanol amine is 1:1-1.4.
A kind of GST purified product, the product include the GST that above-mentioned GST purified material or preparation method are prepared
Purified material；
Preferably, the product includes affinity column or purification kit.
GST purified material that above-mentioned GST purified material or preparation method are prepared or GST purified product are in separation, pure
Change, the application in analysis or identification GST albumen or gst fusion protein；
Preferably, the gst fusion protein includes expressing in mammalian cell, insect cell or Microbial Expression Systems
The label containing GST fusion protein.
Compared with prior art, the invention has the benefit that
GST purified material provided by the invention with solid phase carrier is connect by solid phase carrier, linking arm and by linking arm
Aglucon composition, wherein linking arm includes at least one of hexanediol diglycidyl ether or butanediol diglycidyl ether.It should
GST purified material is optimized by repetition test, meticulously screening raw material, the product prepared.Linking arm is suitable due to having
Molecular chain length and performance specifically bound so that GST is more advantageous to aglucon, improve the purification efficiency of material.
The GST purified material is not only of fine quality but also inexpensive, compared with the similar product of current commercial type, has higher carrying capacity,
Absorption carrying capacity is big, and non-specific adsorption is few, and purification effect is good, while applied widely, is suitble to large-scale device application.
The preparation method of GST purified material provided by the invention is the life that Optimizing Process Parameters obtain by repetition test
Production. art.This method great advantage is that preparation process is simple, low in cost, and performance is stablized, and is suitble to large-scale production preparation.
GST purified product provided by the invention has the advantage of above-mentioned GST purified material, especially good same of purification effect
When it is at low cost, GST purified material can reuse, and further decrease purifying cost.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of GST purified material of the present invention；
The SDS-PAGE result for the GST purified material purification effect detection that Fig. 2 is embodiment 5-7 in the embodiment of the present invention 9
Figure；
Fig. 3 is the SDS-PAGE result figure of contrast product purification effect detection in the embodiment of the present invention 9；
Fig. 4 is that recycling GST purified material purification efficiency changes schematic diagram in the embodiment of the present invention 10.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.
A kind of GST purified material, by solid phase carrier, linking arm and the aglucon group being connect by linking arm with solid phase carrier
At, wherein linking arm includes in hexanediol diglycidyl ether, neopentyl glycol diglycidyl glycerin ether or butanediol diglycidyl ether
At least one, preferably hexanediol diglycidyl ether.
The structural schematic diagram of GST purified material is as shown in Figure 1.The GST purified material is optimized by repetition test, meticulously
Screen raw material, the product prepared.Linking arm is due to suitable molecular chain length and performance, so that GST is more advantageous to
It is specifically bound with aglucon, improves the purification efficiency of material.By the study found that hexanediol diglycidyl ether effect
Fruit is more preferable.The GST purified material is not only of fine quality but also inexpensive, compared with the similar product of current commercial type, has higher
Carrying capacity, absorption carrying capacity it is big, non-specific adsorption is few, and purification effect is good, while applied widely, and suitable large-scale device is answered
With.
In certain embodiments of the present invention, solid phase carrier includes silica gel, controlled pore glass, sephadex, fine jade
At least one of sepharose or cellulose, preferably at least one of Ago-Gel or sephadex, it is further excellent
It is selected as at least one of agarose 4FF, agarose 6FF, agarose CL-4B or agarose CL-6B, further
Preferably agarose 4FF.
Aglucon is fixed on to the silica gel of spherical, unformed, film or threadiness, controlled pore glass, Portugal by linking arm
On polysaccharide gel, Ago-Gel or cellulose, affinitive material can be obtained and prepare corresponding purified product.It is preferable to use
Agarose 4FF stablizes since the granular size of microballoon is between 50-160 μm, and within the scope of the wide pH of 2-12, has
The 4% suitable degree of cross linking, both ensure that the characteristic of the high carrying capacity of product, more have high pressure resistant, high flow rate characteristic, it is suitble to extensive dress
Column application.
In the present invention, one is preferably carried out in mode, and aglucon is GSH albumen.GST can and glutathione
Sulfydryl in (glutathione, GSH) occurs specific covalent and combines, and using this property, GSH can be coupled to solid phase load
On body, and then gst fusion protein is separated from crude extract.Since the combination of GST and GSH has high specificity, because
The fusion protein purity with higher that this its purifying obtains.
A kind of preparation method of GSH purified material, preparation method include the steps that on ligand cou to linking arm,
In, linking arm is connect with solid phase carrier.The preparation method is the production technology that Optimizing Process Parameters obtain by repetition test.
This method great advantage is that preparation process is simple, low in cost, and performance is stablized, and is suitble to large-scale production preparation.
In certain embodiments of the present invention, the step of coupling includes: by aglucon, catalyst and consolidating with linking arm
Phase carrier carries out incubation coupling in PBS solution.Under the effect of the catalyst, dehydration condensation occurs for aglucon and linking arm,
Form GST purified material.The condition of the coupling step is by a large amount of experimental study, and verifying obtains repeatedly, is conducive to aglucon
With the coupling reaction of linking arm, the purification performance of GST purified material is further increased.
In certain embodiments of the present invention, be incubated for coupling condition include: temperature be 45-55 DEG C under the conditions of,
100-150rpm/min is incubated for 12-20h.The reaction condition is obtained by repetition test, and coupling effect is good under this condition, material
Expect that performance is stablized.Typical but non-limiting temperature is 45 DEG C, 47 DEG C, 47 DEG C, 50 DEG C, 52 DEG C, 54 DEG C or 55 DEG C；Revolving speed is typical
But unrestricted is 100rpm/min, 110rpm/min, 120rpm/min, 130rpm/min, 140rpm/min or 150rpm/
min；It is 12 that incubation time is typical but non-limiting, 13h, 14h, 15h, 16h, 17h, 18h, 19h or 20h.
In certain embodiments of the present invention, the concentration of PBS solution is 10-30mM.The concentration of PBS solution is typical but non-
Restrictive is 10mM, 15mM, 20mM, 25mM or 30mM.
In certain embodiments of the present invention, the volume ratio of the solid phase carrier with linking arm and PBS solution is 1:1-
2.The typical but non-limiting volume ratio of solid phase carrier and PBS solution with linking arm is 1:1,1:1.2,1:1.4,1:
1.6,1:1.8 or 1:2.It should be noted that the solid phase carrier volume in the application refers to settling volume.
In certain embodiments of the present invention, the concentration of aglucon is 5.3-5.9g/L.The concentration of aglucon is typical but non-limit
Property processed is 5.3g/L, 5.4g/L, 5.5g/L, 5.6g/L, 5.7g/L, 5.8g/L or 5.9g/L.
In certain embodiments of the present invention, catalyst includes NaBH4Or KBH4, preferably NaBH4, further,
NaBH4Concentration be 1-1.4g/L.NaBH4Concentration it is typical but non-limiting for 1g/L, 1.1g/L, 1.2g/L, 1.3g/L or
1.4g/L。
It optimizes and limits by the proportion to reaction raw materials, can further guarantee the performance of GST purified material
And purification effect.
The step of one is preferably carried out in mode, and linking arm is connect with solid phase carrier in the present invention includes: by linking arm
Under alkaline condition with solid phase carrier, incubation connection is carried out by catalyst.Under the specific alkaline environment, by solid phase carrier
Upper exposed hydroxyl (- OH) activation, under the catalytic action of catalyst, linking arm and the hydroxyl on solid phase carrier are passed through
The form of chemical bond connects, and the obtained solid phase carrier property for being connected with linking arm is stablized, not easily to fall off therebetween.
In certain embodiments of the present invention, the volume ratio of solid phase carrier and linking arm is 1:0.4-0.8.Solid phase carrier
Typical but non-limiting with the volume ratio of linking arm is 1:0.4,1:0.5,1:0.6,1:0.7 or 1:0.8.
In certain embodiments of the present invention, the condition for being incubated for connection includes: 100- under the conditions of 35-45 DEG C
150rpm/min is incubated for 5-11h.The reaction condition is obtained by repetition test, under this condition good connecting effect, material
It can stablize.Typical but non-limiting temperature is 35 DEG C, 37 DEG C, 39 DEG C, 40 DEG C, 42 DEG C, 44 DEG C or 45 DEG C；Revolving speed is typical but non-
Restrictive is 100rpm/min, 110rpm/min, 120rpm/min, 130rpm/min, 140rpm/min or 150rpm/min；
Typical but non-limiting incubation time is 5h, 6h, 7h, 8h, 9h, 10h or 11h.
In certain embodiments of the present invention, alkaline condition includes the organic solution containing NaOH or KOH, is preferably contained
There is the organic solution of NaOH, further, the concentration of NaOH is preferably 25-35g/L.The concentration of NaOH is typical but non-limiting
For 25g/L, 27g/L, 29g/L, 30g/L, 32g/L, 34g/L or 35g/L.
In certain embodiments of the present invention, organic solution includes dimethyl sulfoxide or dimethylformamide, preferably
Dimethyl sulfoxide.Under the instrumentality of dimethyl sulfoxide or dimethylformamide, the hydroxyl on solid phase carrier is obtained adequately
Activation is conducive to subsequent connect reaction with linking arm.
In certain embodiments of the present invention, the volume ratio of solid phase carrier and dimethyl sulfoxide is 1:1.2-1.6.Solid phase
The typical but non-limiting volume ratio of carrier and dimethyl sulfoxide is 1:1.2,1:1.6 or 1:1.4.
In some embodiments of the present invention, catalyst includes NaBH4Or KBH4, preferably NaBH4, further,
NaBH4Concentration be preferably 1-3g/L.NaBH4Concentration it is typical but non-limiting for 1g/L, 1.5g/L, 2g/L, 2.5g/L or
3g/L。
It optimizes and limits by the proportion to reaction raw materials, further ensure what linking arm and solid phase carrier combined
Stability.
In the present invention, one is preferably carried out in mode, and linking arm includes hexanediol diglycidyl ether, the contracting of pentanediol two
At least one of water glycerin ether or butanediol diglycidyl ether, preferably hexanediol diglycidyl ether.Hexylene glycol two contracts
The molecular chain length of water glycerin ether and butanediol diglycidyl ether is suitable, is conducive to the specific binding of GST and aglucon.
In the present invention, one is preferably carried out in mode, and solid phase carrier includes that silica gel, controlled pore glass, macromolecule are micro-
In at least one of ball, sephadex, Ago-Gel or cellulose, preferably Ago-Gel or sephadex extremely
Few one kind, further preferably agarose 4FF, agarose 6FF, in agarose CL-4B or agarose CL-6B extremely
Few one kind, is still more preferably agarose 4FF.It is preferable to use agarose 4FF, since the granular size of microballoon is in 50-
Between 160 μm, and stablize within the scope of the wide pH of 2-12, with the 4% suitable degree of cross linking, both ensure that the high carrying capacity of product
Characteristic more has high pressure resistant, high flow rate characteristic, is suitble to extensive dress column application.
In the present invention, one is preferably carried out in mode, and aglucon is GSH albumen.GST can and glutathione
Sulfydryl in (glutathione, GSH) occurs specific covalent and combines, and using this property, GSH can be coupled to solid phase load
On body, and then gst fusion protein is separated from crude extract.Since the combination of GST and GSH has high specificity, because
The fusion protein purity with higher that this its purifying obtains.
It in certain embodiments of the present invention, further include the step that solid phase carrier deactivates after aglucon and linking arm are coupled
Suddenly, wherein the step of deactivating includes: to be closed the site not connecting with linking arm on solid phase carrier with ethanol amine.
In certain embodiments of the present invention, closed condition includes: 100-150rpm/ under the conditions of 35-40 DEG C
Min closes 12-20h.To not there is no the active group for connecting linking arm to close on solid phase carrier, advantageously reduces non-specificity
Absorption, improves the efficiency of purifying.Typical but non-limiting temperature is 35 DEG C, 36 DEG C, 37 DEG C, 38 DEG C, 39 DEG C or 40 DEG C；Revolving speed
It is typical but non-limiting for 100rpm/min, 110rpm/min, 120rpm/min, 130rpm/min, 140rpm/min or
150rpm/min；It is 12 that off-period is typical but non-limiting, 13h, 14h, 15h, 16h, 17h, 18h, 19h or 20h.
It is preferably carried out in mode at of the invention one, the solvent to deactivate is 3-7mM EDTA.
It is preferably carried out in mode at of the invention one, the concentration of ethanol amine is 0.5-1.5M.
It is preferably carried out in mode at of the invention one, the volume ratio of solid phase carrier and ethanol amine is 1:1-1.4.Solid phase
The typical but non-limiting volume ratio of carrier and ethanol amine is 1:1,1:1.1,1:1.2,1:1.3 or 1:1.4.
By to the proportion of component used and the optimization of reaction condition and restriction of deactivating, so that not connected linking arm is consolidated
Phase carrier is effectively deactivated, and non-specific adsorption is effectively prevented, and improves the performance and purifying effect of GST purified material
Fruit.
In the present invention, one is preferably carried out in mode, the preparation method of GST purified material the following steps are included:
A solid carrier) is taken, vacuum filtration machine is drained, and is dipped in deionized water and is impregnated no less than 6h, drains, repeatedly for three times；
B it) is added into the solid phase carrier drained, the dimethyl sulfoxide of 1.2-1.6 times of solid phase carrier volume, 0.4-0.8 times
The hexanediol diglycidyl ether of solid phase carrier volume mixes, and NaOH, NaBH of 0.1 times of solid phase carrier volume is then added4It is mixed
Conjunction solution (mother liquid concentration 10 ×, i.e. NaOH is 300g/L, NaBH4For 20g/L, similarly hereinafter), until the final concentration of 30g/L of NaOH,
The final concentration of 2g/L of NaBH4, is finally placed in 35-45 DEG C of constant-temperature table, and 100-150rpm is incubated for 5-11h；
C) after reaction, drain, be dipped in deionized water and impregnate no less than 6h, drain, repeatedly for three times；
D the 20mM PBS that 1-3 times of volume) is added into the solid phase carrier with hexanediol diglycidyl ether drained is molten
Liquid mixes, aglucon, NaBH4 powder is then respectively adding, until the final concentration of 5.3-5.9g/L, NaBH of aglucon4Final concentration of 1-
1.4g/L, is finally placed in 45-55 DEG C of constant-temperature table, and 100-150rpm is incubated for 12-20h；
E) after reaction, drain, be dipped in deionized water and impregnate no less than 6h, drain, repeatedly for three times；
F the 1M ethanolamine solutions of 1-1.4 times of agarose volume) are added into the solid phase carrier drained, mixes, is then added
EDTA solution (mother liquid concentration 500mM) is finally placed in 35-40 DEG C of constant-temperature table, 100-150rpm is incubated for 12- to final concentration 5mM
20h；
G) after reaction, drain, be dipped in deionized water and impregnate no less than 6h, drain, repeatedly for three times, be finally stored in
1 × PBS, pH7.4,20% ethanol solution.
The GST purified material that above-mentioned preparation method is prepared has single-minded active force strong, and raw material is easy to get, cheap same
When the good feature of purification effect.
A kind of GST purified material that GST purified product is prepared comprising above-mentioned GST purified material or preparation method.It produces
Product have the advantage of above-mentioned GST purified material, and at low cost while especially purification effect is good, GST purified material can repeat
It utilizes, further decreases purifying cost.
It is preferably carried out in mode in the present invention, product includes affinity column or purification kit.
GST purified material that above-mentioned GST purified material or preparation method are prepared or GST purified product are in separation, pure
Change, the application in analysis or identification GST albumen or gst fusion protein.GST purified material provided by the invention can be applied to respectively
In the relevant field of kind GST albumen, reaction environment can be liquid phase or solid phase, and reaction volume can be micro or high-volume, produce
Moral character can be good, and purification efficiency is high, is suitble to various application scenarios.
In certain embodiments of the present invention, gst fusion protein includes mammalian cell, insect cell or microorganism
The fusion protein for the label containing GST expressed in expression system.
Present invention will be further explained by specific examples below, it should be understood, however, that, these embodiments are only
It is used, is but should not be understood as present invention is limited in any form for being described in more detail.
Embodiment 1
The present embodiment provides a kind of preparation methods of GST purified material, comprising the following steps:
A) go bail for the agarose agarose 6FF being stored in 20% ethyl alcohol, vacuum filtration machine is drained, is dipped in deionized water
It impregnates and is no less than 6h, drain, repeatedly for three times；
B the dimethyl sulfoxide of 1.2 times of agarose volumes, 0.8 times of agarose volume) are added into the agarose particle drained
Butanediol diglycidyl ether, mix, then be added 0.1 times of agarose volume NaOH, NaBH4(mother liquor is dense for mixed solution
Degree 10 ×, i.e. NaOH be 300g/L, NaBH4 20g/L, similarly hereinafter), until the final concentration of 2g/ of NaOH final concentration of 30g/L, NaBH4
L, is finally placed in 35 DEG C of constant-temperature tables, and 100-150rpm is incubated for 11h；
C) after reaction, drain, be dipped in deionized water and impregnate no less than 6h, drain, repeatedly for three times；
D the 20mM PBS solution of 1 times of agarose volume) is added into the agarose particle drained, mixes, then adds respectively
Enter GSH, NaBH4Powder, until GSH final concentration of 5.9g/L, NaBH4Final concentration of 1g/L is finally placed in 55 DEG C of constant-temperature tables,
100-150rpm is incubated for 12h；
E) after reaction, drain, be dipped in deionized water and impregnate no less than 6h, drain, repeatedly for three times；
F the 1M ethanolamine solutions of 1.4 times of agarose volumes) are added into the agarose particle drained, mixes, is then added
EDTA solution (mother liquid concentration 500mM) is finally placed in 35 DEG C of constant-temperature tables, 100-150rpm is incubated for 20h to final concentration 5mM；
G) after reaction, drain, be dipped in deionized water and impregnate no less than 6h, drain, repeatedly for three times, be finally stored in
1 × PBS, pH7.4,20% ethanol solution.
Embodiment 2
The present embodiment provides a kind of preparation methods of GST purified material, comprising the following steps:
A) go bail for the agarose agarose CL-4B being stored in 20% ethyl alcohol, vacuum filtration machine is drained, and is dipped in deionized water
Middle impregnate is no less than 6h, drains, repeatedly for three times；
B the dimethyl sulfoxide of 1.6 times of agarose volumes, 0.4 times of agarose volume) are added into the agarose particle drained
Neopentyl glycol diglycidyl glycerin ether, mix, then be added 0.1 times of agarose volume NaOH, NaBH4Mixed solution, until NaOH
The final concentration of 2g/L of final concentration of 30g/L, NaBH4, is finally placed in 45 DEG C of constant-temperature tables, and 100-150rpm is incubated for 5h；
C) after reaction, drain, be dipped in deionized water and impregnate no less than 6h, drain, repeatedly for three times；
D the 20mM PBS solution of 2 times of agarose volumes) is added into the agarose particle drained, mixes, then adds respectively
Enter GSH, NaBH4Powder, until GSH final concentration of 5.3g/L, NaBH4Final concentration of 1.4g/L is finally placed in 45 DEG C of constant-temperature tables,
100-150rpm is incubated for 20h；
E) after reaction, drain, be dipped in deionized water and impregnate no less than 6h, drain, repeatedly for three times；
F the 1M ethanolamine solutions of 1 times of agarose volume) are added into the agarose particle drained, mixes, is then added
EDTA solution (mother liquid concentration 500mM) is finally placed in 40 DEG C of constant-temperature tables, 100-150rpm is incubated for 12h to final concentration 5mM；
G) after reaction, drain, be dipped in deionized water and impregnate no less than 6h, drain, repeatedly for three times, be finally stored in
1 × PBS, pH7.4,20% ethanol solution.
Embodiment 3
The present embodiment provides a kind of preparation methods of GST purified material, comprising the following steps:
A) go bail for the agarose agarose CL-6B being stored in 20% ethyl alcohol, vacuum filtration machine is drained, and is dipped in deionized water
Middle impregnate is no less than 6h, drains, repeatedly for three times；
B the dimethyl sulfoxide of 1.4 times of agarose volumes, 0.6 times of agarose volume) are added into the agarose particle drained
Butanediol diglycidyl ether, mix, then be added 0.1 times of agarose volume NaOH, NaBH4Mixed solution, until NaOH
The final concentration of 2g/L of final concentration of 30g/L, NaBH4, is finally placed in 40 DEG C of constant-temperature tables, and 100-150rpm is incubated for 8h；
C) after reaction, drain, be dipped in deionized water and impregnate no less than 6h, drain, repeatedly for three times；
D the 20mM PBS solution of 1.5 times of agarose volumes) is added into the agarose particle drained, mixes, then distinguishes
GSH, NaBH is added4Powder, until GSH final concentration of 5.6g/L, NaBH4Final concentration of 1.2g/L is finally placed in 50 DEG C of constant temperature and shakes
Bed, 100-150rpm are incubated for 16h；
E) after reaction, drain, be dipped in deionized water and impregnate no less than 6h, drain, repeatedly for three times；
F the 1M ethanolamine solutions of 1.2 times of agarose volumes) are added into the agarose particle drained, mixes, is then added
EDTA solution (mother liquid concentration 500mM) is finally placed in 37 DEG C of constant-temperature tables, 100-150rpm is incubated for 16h to final concentration 5mM；
G) after reaction, drain, be dipped in deionized water and impregnate no less than 6h, drain, repeatedly for three times, be finally stored in
1 × PBS, pH7.4,20% ethanol solution.
Embodiment 4
The present embodiment provides a kind of preparation methods of GST purified material, comprising the following steps:
A) go bail for the agarose agarose 4FF being stored in 20% ethyl alcohol, vacuum filtration machine is drained, is dipped in deionized water
It impregnates and is no less than 6h, drain, repeatedly for three times；
B the dimethyl sulfoxide of 1.4 times of agarose volumes, 0.6 times of agarose volume) are added into the agarose particle drained
Hexanediol diglycidyl ether, mix, then be added 0.1 times of agarose volume NaOH, NaBH4Mixed solution, until NaOH
The final concentration of 2g/L of final concentration of 30g/L, NaBH4, is finally placed in 40 DEG C of constant-temperature tables, and 100-150rpm is incubated for 8h；
C) after reaction, drain, be dipped in deionized water and impregnate no less than 6h, drain, repeatedly for three times；
D the 20mM PBS solution of 1.5 times of agarose volumes) is added into the agarose particle drained, mixes, then distinguishes
GSH, NaBH is added4Powder, until GSH final concentration of 5.6g/L, NaBH4Final concentration of 1.2g/L is finally placed in 50 DEG C of constant temperature and shakes
Bed, 100-150rpm are incubated for 16h；
E) after reaction, drain, be dipped in deionized water and impregnate no less than 6h, drain, repeatedly for three times；
F the 1M ethanolamine solutions of 1.2 times of agarose volumes) are added into the agarose particle drained, mixes, is then added
EDTA solution (mother liquid concentration 500mM) is finally placed in 37 DEG C of constant-temperature tables, 100-150rpm is incubated for 16h to final concentration 5mM；
G) after reaction, drain, be dipped in deionized water and impregnate no less than 6h, drain, repeatedly for three times, be finally stored in
1 × PBS, pH7.4,20% ethanol solution.
The preparation of 5 lab scale of embodiment
Activation: 10mL agarose 4FF agarose particle, washing are added in 50mL conical flask after draining, sequentially add
14mL dimethyl sulfoxide, 6mL hexanediol diglycidyl ether, 1mL NaOH, NaBH4Mother liquor, mixing are placed on 40 DEG C of constant temperature and shake
Bed, 100-150rpm are incubated for 8h.
Coupling: agarose particle washing is added in 50mL conical flask after draining, and it is molten to sequentially add 15mL 20mM PBS
Liquid, 56mg GSH, 12mg NaBH4, mix and be placed on 50 DEG C of constant-temperature tables, 100-150rpm is incubated for 16h.
Deactivate: agarose particle washing is added in 50mL conical flask after draining, and it is molten to sequentially add 12mL ethanol amine
Liquid, 0.2mL EDTA mother liquor, mixing are placed on 37 DEG C of constant-temperature tables, and 100-150rpm is incubated for 16h.
It saves: being stored in 1 × PBS, pH7.4,20% ethanol solution after washing.
6 pilot scale of embodiment preparation
Activation: 100mL agarose 4FF agarose particle, washing drain after be added 500mL conical flask in, successively plus
Enter 140mL dimethyl sulfoxide, 60mL hexanediol diglycidyl ether, 10mLNaOH, NaBH4Mother liquor, mixing are placed on 40 DEG C of perseverances
Warm shaking table, 100-150rpm are incubated for 8h.
Coupling: agarose particle washing is added in 500mL conical flask after draining, and sequentially adds 150mL 20mM PBS
Solution, 0.56g GSH, 0.12g NaBH4, mix and be placed on 50 DEG C of constant-temperature tables, 100-150rpm is incubated for 16h.
Deactivate: agarose particle washing is added in 500mL conical flask after draining, and sequentially adds 120mL ethanol amine
Solution, 2mL EDTA mother liquor, mixing are placed on 37 DEG C of constant-temperature tables, and 100-150rpm is incubated for 16h.
It saves: being stored in 1 × PBS, pH7.4,20% ethanol solution after washing.
Embodiment 7 is manufactured experimently greatly standby
Activation: 1L agarose 4FF agarose particle, washing are added in 5L conical flask after draining, and sequentially add 1.4L
Dimethyl sulfoxide, 0.6L hexanediol diglycidyl ether, 0.1L NaOH, NaBH4Mother liquor, mixing are placed on 40 DEG C of constant-temperature tables,
100-150rpm is incubated for 8h.
Coupling: agarose particle washing is added in 5L conical flask after draining, and it is molten to sequentially add 1.5L 20mM PBS
Liquid, 5.6g GSH, 1.2g NaBH4, mix and be placed on 50 DEG C of constant-temperature tables, 100-150rpm is incubated for 16h.
Deactivate: agarose particle washing is added in 5L conical flask after draining, and sequentially adds 1.2L ethanolamine solutions,
20mL EDTA mother liquor, mixing are placed on 37 DEG C of constant-temperature tables, and 100-150rpm is incubated for 16h.
It saves: being stored in 1 × PBS, pH7.4,20% ethanol solution after washing.
Embodiment 8 induces Bacillus coli expression GST-Mut fusion protein
A it) goes bail for the strain deposited, is inoculated in 100mL LB culture medium (ampicillin containing 50mg/ml) by 1:1000,37
DEG C, 220rpm activation is overnight；
B activated strain) is taken, is inoculated in 3L LB culture medium (ampicillin containing 50mg/ml) in 1:100 ratio,
37 DEG C, 220rpm culture；
C OD) is arrived600When reaching 0.6 or so, stop culture, bacterium solution temperature is down to 20 DEG C, IPTG is added to final concentration
0.2mM, 20 DEG C, 220rpm overnight incubation；
D) culture finishes, and cell is collected by centrifugation.
The purification effect of embodiment 9GST purified material detects
1. the cell being collected into embodiment 8 being blown and beaten with 150mL Lysis Buffer, suspension, ultrasonication being made
Afterwards, 4 DEG C, 12000rpm is centrifuged 20min, isolates that cell cracking supernatant is spare, and precipitating is given up；
2. the GST purified material of embodiment 5-7 preparation respectively takes 1mL, it is separately added into corresponding chromatographic column, Binding
Buffer balancing material carries out affinity chromatography experiment respectively；
3. each material is separately added into 50mL cell cracking supernatant, 4 DEG C of shaking tables are incubated for 1h；
4. samples of incubation is added in chromatographic column, collection material, Binding Buffer balances 10CV (column
volume)；
5.Elution Buffer elutes 5CV；
6. each component to get off is washed in collection, loading sample is made in each loading buffer that 1CV is added, and SDS-PAGE is solidifying
Gel electrophoresis, as a result as shown in Fig. 2, wherein M:protein marker；1: sample solution；2: flowing through liquid；3: elution；4: resin
Residual；
Note: Lysis Buffer/Binding Buffer:1 × PBS, 20%Glycerol, pH7.4；
Elution Buffer:1 × PBS, 20%Glycerol, 20mM GSH, pH7.4.
The protein concentration of elution fraction after GST-Mut affinity chromatography, by non-interference type quantification of protein kit measurement
(raw work biological product #C503071), and thus calculate the carrying capacity of material.
With XXX product, product is detected as a comparison for the purification effect detection test of GST purified material, contrast product
PAGE gel electrophoresis, as a result as shown in Fig. 3, wherein M:protein marker；1: sample solution；2: flowing through liquid；3: washing
It is de-；4: resin residue；
The carrying capacity of embodiment 5-7 and the material of contrast product, non-specific adsorption and material residual quantity are counted, tied
Fruit is as follows:
| Embodiment 5 | Embodiment 6 | Embodiment 7 | Contrast product | |
| Carrying capacity mg/ml | 12.6 | 11.5 | 13.6 | 9.7 |
| Non-specific adsorption | Seldom | Seldom | Seldom | It is less |
| Resin residue | Seldom | Seldom | Seldom | It is less |
Embodiment 10GST purified material reuses effect detection
After GST label Purification Resin prepared by embodiment 7 is purified GST-Mut by embodiment 9, according to the following steps clearly
It washes:
1. deionized water rinses 10CV；
2.8M Urea, 1 × PBS, pH7.4 rinse 10CV；
3. deionized water rinses 10CV；
4.0.1M NaAc, 0.5M NaCl, 10mM EDTA, pH4.0 rinse 10CV；
5. deionized water rinses 10CV；
6.20% ethyl alcohol, 1 × PBS are saved.
After recycling resin, 9 same method of embodiment tests GST purified material purification effect, SDS-PAGE analysis, recycling 6
It remains to reach very high purification efficiency after secondary, as a result as shown in Figure 4.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention
Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (10)
1. a kind of GST purified material, which is characterized in that connect including solid phase carrier, linking arm and by linking arm and solid phase carrier
The aglucon connect；
The linking arm includes in hexanediol diglycidyl ether, neopentyl glycol diglycidyl glycerin ether or butanediol diglycidyl ether
At least one.
2. GST purified material according to claim 1, which is characterized in that the solid phase carrier includes silica gel, controllable bore diameter
In at least one of glass, sephadex, Ago-Gel or cellulose, preferably Ago-Gel or sephadex
At least one, further preferably agarose 4FF, agarose 6FF, in agarose CL-4B or agarose CL-6B
At least one is still more preferably agarose 4FF.
3. GST purified material according to claim 1 or 2, which is characterized in that the aglucon is GSH albumen.
4. a kind of preparation method of GSH purified material, which is characterized in that the preparation method includes by ligand cou to linking arm
On step, wherein the linking arm is connect with solid phase carrier.
5. the preparation method according to claim 4, which is characterized in that the step of coupling includes: by aglucon, catalyst
Incubation coupling is carried out in PBS solution with the solid phase carrier with linking arm；
Preferably, the condition for being incubated for coupling includes: under the conditions of temperature is 45-55 DEG C, and 100-150rpm/min is incubated for 12-
20h；
Preferably, the concentration of the PBS solution is 10-30mM；
Preferably, the volume ratio of the solid phase carrier with linking arm and the PBS solution is 1:1-2；
Preferably, the concentration of the aglucon is 5.3-5.9g/L；
Preferably, the catalyst includes NaBH4Or KBH4, preferably NaBH4；
Preferably, the NaBH4Concentration be 1-1.4g/L.
6. the preparation method according to claim 4, which is characterized in that the step of linking arm is connect with solid phase carrier is wrapped
It includes: under alkaline condition by linking arm and solid phase carrier, incubation connection being carried out by catalyst；
Preferably, the volume ratio of the solid phase carrier and the linking arm is 1:0.4-0.8；
Preferably, the condition for being incubated for connection includes: under the conditions of 35-45 DEG C, and 100-150rpm/min is incubated for 5-11h；
Preferably, the alkaline condition includes the organic solution containing NaOH or KOH, preferably containing the organic solution of NaOH；
Preferably, the concentration of NaOH is 25-35g/L；
Preferably, the organic solution includes dimethyl sulfoxide or dimethylformamide, preferably dimethyl sulfoxide；
Preferably, the volume ratio of the solid phase carrier and the dimethyl sulfoxide is 1:1.2-1.6；
Preferably, the catalyst includes NaBH4Or KBH4, preferably NaBH4；
Preferably, NaBH4Concentration be 1-3g/L.
7. the preparation method according to claim 4, which is characterized in that the linking arm includes hexylene glycol 2-glycidyl
At least one of ether, neopentyl glycol diglycidyl glycerin ether or butanediol diglycidyl ether；
Preferably, the solid phase carrier includes in silica gel, controlled pore glass, sephadex, Ago-Gel or cellulose
At least one, preferably at least one of Ago-Gel or sephadex, further preferably agarose 4FF,
At least one of agarose 6FF, agarose CL-4B or agarose CL-6B are still more preferably agarose
4FF；
Preferably, the aglucon is GSH albumen.
8. according to the described in any item preparation methods of claim 4-7, which is characterized in that further include after aglucon and linking arm coupling
The step of solid phase carrier deactivates；
Described the step of deactivating includes: to be closed the site not connecting with linking arm on solid phase carrier with ethanol amine；
Preferably, the closed condition includes: under the conditions of 35-40 DEG C, and 100-150rpm/min closes 12-20h；
Preferably, the solvent to deactivate is 3-7mM EDTA；
Preferably, the concentration of the ethanol amine is 0.5-1.5M；
Preferably, the volume ratio of the solid phase carrier and the ethanol amine is 1:1-1.4.
9. a kind of GST purified product, which is characterized in that the product includes that the described in any item GST of claim 1-3 purify material
The GST purified material that material or the described in any item preparation methods of claim 4-8 are prepared；
Preferably, the product includes affinity column or purification kit.
10. the described in any item GST purified materials of claim 1-3 or the described in any item preparation method systems of claim 4-8
Standby obtained GST purified material or GST purified product as claimed in claim 9 are in separation, purifying, analysis or identification GST albumen
Or the application in gst fusion protein；
Preferably, the gst fusion protein includes containing of expressing in mammalian cell, insect cell or Microbial Expression Systems
The fusion protein of GST label.
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