阅读说明：本技术 重组人金属硫蛋白ⅲ纯品的制备方法 (Preparation method of recombinant human metallothionein III pure product ) 是由 胡小垒 原海亮 秦建新 冯永良 于 2019-11-28 设计创作，主要内容包括：一种重组人金属硫蛋白Ⅲ纯品的制备方法,属于基因工程重组蛋白质纯化技术领域。步骤：制备融合蛋白溶液；制备上清液Ⅱ；制备酶切液；制备MT-III蛋白的粗品液；制备含MT-III蛋白的半成品溶液；收集巯基峰洗脱液；将巯基峰洗脱液进行超滤除盐,然后冷冻干燥成冻干粉,得到重组人金属硫蛋白Ⅲ纯品。优点：在获得高纯度重组人金属硫蛋白Ⅲ的同时,避免使用昂贵的GST亲和层析介质,降低制备成本；既可促进金属硫蛋白空间结构的正确折叠,防止金属硫蛋白片段聚合析出；使得获得的重组人金属硫蛋白III纯度及稳定性更好。(preparation method of pure recombinant human metallothionein III, belonging to the technical field of gene engineering recombinant protein purification, comprises the steps of preparing fusion protein solution, preparing supernatant II, preparing enzyme digestion solution, preparing crude product solution of MT-III protein, preparing semi-finished product solution containing MT-III protein, collecting sulfydryl peak eluent, carrying out ultrafiltration desalination on the sulfydryl peak eluent, and then carrying out freeze drying to obtain pure recombinant human metallothionein III.)

1, kinds of recombinant human metallothionein III pure product preparation method, characterized by comprising the following steps:

A) preparing a fusion protein solution, namely resuspending the recombinant human metallothionein III fermentation thalli in a buffer solution I, then carrying out ultrasonic crushing, then carrying out centrifugal separation, and collecting a supernatant I to obtain a fusion protein solution containing GST-MT-III;

B) preparing a crude fusion protein GST-MT-III product, carrying out fractional precipitation on the supernatant I obtained in the step A) by using ammonium sulfate, and controlling the technological parameters of the fractional precipitation to obtain the crude fusion protein GST-MT-III product;

C) preparing a supernatant II, namely resuspending the fusion protein GST-MT-III crude product obtained in the step B) in a buffer II to obtain a resuspension solution, adjusting the pH value of the resuspension solution by using a pH regulator, and then performing centrifugal separation to remove insoluble impurity proteins to obtain the supernatant II;

D) preparing enzyme digestion solution, firstly carrying out ultrafiltration on the supernatant II obtained in the step C) by using an ultrafiltration membrane and concentrating to obtain concentrated solution, then adjusting the pH value of the concentrated solution, then adding zinc sulfate and thrombin, and stirring for reaction to obtain the enzyme digestion solution;

E) preparing a crude product liquid of the MT-III protein, heating the enzyme digestion liquid obtained in the step D) to thermally denature and precipitate impurity protein, and performing centrifugal separation to obtain a crude product liquid of the MT-III protein;

F) preparing a semi-finished product solution containing MT-III protein, adjusting the conductivity of the crude product solution of MT-III protein obtained in the step E) until the conductivity of the crude product solution of MT-III protein is equivalent to that of the buffer solution III to obtain the crude product solution of MT-III protein after the conductivity is adjusted, then loading the crude product solution of MT-III protein after the conductivity is adjusted on a hydrophobic chromatographic column which is well balanced in advance, and collecting penetrating liquid to obtain the semi-finished product solution containing MT-III protein;

G) collecting sulfydryl peak eluent, carrying out ultrafiltration desalination on the semi-finished product solution of the MT-III protein obtained in the step F) by using an ultrafiltration membrane, then loading the semi-finished product solution on a well-balanced anion exchange chromatographic column, carrying out gradient elution by using a buffer solution IV and a buffer solution V, and collecting the eluent by separating bottles to obtain sulfydryl peak eluent;

H) and G) preparing a finished product, namely performing ultrafiltration desalting on the sulfydryl peak eluent obtained in the step G), and then freeze-drying the sulfydryl peak eluent into lyophilized powder to obtain a pure recombinant human metallothionein III product.

2. The method for preparing the pure recombinant human metallothionein III according to claim 1, wherein the volume-to-mass ratio of the buffer solution I and the recombinant human metallothionein III fermented bacteria in the step A) is 2-5: 1.

3. The method for preparing purified recombinant human metallothionein III according to claim 1 or 2, wherein the buffer solution I is or more selected from phosphate buffer solution, Tris-HCl buffer solution and TEA buffer solution with pH value of 5-9.

4. The method of claim 1, wherein said step B) of fractionating said supernatant I with ammonium sulfate comprises -grade and two-grade precipitations, and said process parameters for controlling fractionation comprise -grade process parameters for controlling two-grade precipitation and process parameters for controlling two-grade precipitation.

5. The method for preparing the pure recombinant human metallothionein III according to claim 4, wherein the -grade precipitation control process comprises the steps of firstly adjusting the temperature of the supernatant I, then slowly adding solid ammonium sulfate in a stirring state until the percent saturation of the ammonium sulfate is 20-50%, standing after the ammonium sulfate is completely dissolved to obtain -grade precipitate, and the second-grade precipitation control process comprises the steps of firstly carrying out centrifugal separation on the -grade precipitate, collecting supernatant, then adjusting the temperature of the supernatant, then slowly adding the solid ammonium sulfate in the stirring state to increase the percent saturation of the ammonium sulfate in the supernatant to 60-90%, standing again after the ammonium sulfate is completely dissolved to obtain second-grade precipitate, and then carrying out centrifugal separation.

6. The method for preparing purified recombinant human metallothionein III according to claim 5, wherein the temperature of the supernatant I is adjusted to 20 to 40 ℃, and the standing is performed at 20 to 40 ℃ for 60 to 100 min; the temperature of the supernatant is adjusted to 20-40 ℃, and the secondary standing is performed for 10-15h at 20-40 ℃.

7. The method for preparing purified recombinant human metallothionein III according to claim 1, wherein the buffer solution II in step C) is or more selected from phosphate buffer solution with pH6-9, Tris-HCl buffer solution and TEA buffer solution, and the adjustment of the pH of the resuspension with pH adjusting agent is performed by adjusting the pH of the resuspension to 6-9 with 1mol/L Tris aqueous solution.

8. The method for preparing purified recombinant human metallothionein III according to claim 1, wherein the step D) of ultrafiltration of the supernatant II with an ultrafiltration membrane is that the supernatant II is subjected to ultrafiltration with an ultrafiltration membrane with molecular weight cut-off of 5-20K, and the concentration is that the volume of the supernatant II after ultrafiltration is concentrated by 2-5 times; the pH value of the concentrated solution is adjusted to 6-9 by adopting 1mol/L Tris; the adding amount of the zinc sulfate is that the final concentration of the zinc sulfate in the enzyme digestion solution reaches 0.05-0.2 mmol/L; the thrombin is an enzyme for specifically shearing a GST label, and the addition amount of the thrombin is 10000U per 100g of the recombinant human metallothionein III thallus; the stirring reaction is carried out at the temperature of 20-25 ℃ and at the speed of 50-100rpm for 14-18 h.

9. The method of claim 1, wherein the step E) of heating the digested solution is performed at 50-80 ℃ for 3-10min, the step F) of adjusting the conductivity of the crude MT-III protein solution obtained in step E) is performed by using ammonium sulfate as a reagent, the buffer III is selected from the group consisting of phosphate buffer solution with pH of 6-9 and containing 1mol/L ammonium sulfate, Tris-HCl buffer solution and or more of TEA buffer solution, and the hydrophobic chromatography column is phenyl-agarose gel chromatography column.

10. The method for preparing the purified recombinant human metallothionein III according to claim 1, wherein the ultrafiltration membrane in the step G) is an ultrafiltration membrane with the molecular weight cut-off of 1-3K, the buffer IV is or more of phosphate buffer solution with the pH value of 6-9, Tris-Hcl buffer solution and TEA buffer solution, the anion exchange chromatography column is a DEAE sepharose gel chromatography column, the buffer V is phosphate buffer solution with the pH value of 6-9 and containing NaCl with the final concentration of 0.4mol/L, Tris-Hcl buffer solution and or more of TEA buffer solution, the volume of the sulfhydryl peak eluent is 3-5 times that of the DEAE sepharose gel column chromatography column, the gradient elution is that the buffer IV and the buffer V are mixed and eluted by a binary gradient pump, the gradient ratio is changed, the ratio of the buffer IV is changed from 100% to 0% in the elution time of 2-5 column volumes, the salt ratio of the buffer V is from 0% to 100%, and the desalting amount of the salt in the ultrafiltration membrane in the step V) is 3-5K molecular weight cut-5.

Technical Field

The invention belongs to the technical field of recombinant protein purification in genetic engineering, and particularly relates to a preparation method of recombinant human metallothionein III pure products.

Background

Metallothionein (MT) is type protein rich in cysteine and small in molecular weight, the structure of MT family protein is well conserved, and consists of 60-61 amino acids, about 30% of which is cysteine (Cys) and does not contain disulfide bond, aromatic amino acid and histidine, while Metallothionein III (MT-III) is originally nerve growth inhibitory factor (NGF) found in the study of central nervous lesion mechanism of human, and the MT-III is proved to be highly homologous with the Metallothionein family protein after the study, so the MT-III distribution is mainly limited to central nervous system, mainly distributed in astrocyte (concentrated in cell body and protrusion), secondly is neuron cell, and is reported to be distributed in germ cell, small amount in stomach, kidney and olfactory cortical cell.

Besides being involved in the process of certain diseases, MT can also be involved in the transportation, storage and metabolism of trace metal elements such as Zn, Cu and Co in vivo; participating in the detoxification of heavy metals such as Hg, Cd, Pb and the like in vivo; participating in radiation resistance, and the capability of scavenging free radicals, especially hydroxyl free radicals is more than 100 times stronger than that of SOD.

At present, most of MT in the market is extracted from animal livers (such as rabbit livers), the obtained product is a mixture of MT-I and MT-II, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) detects that most of products are in a dispersed or polymerized state, the single property of the product is poor, and when the protein extracted from animal tissues is used for human bodies, the biosafety problem exists, meanwhile, a large number of animals are needed as production raw materials for industrial production, and the ecological friendliness is poor.

In the current genetic engineering protein research, the purification of recombinant protein mostly depends on specific tags (such as GST tags, His tags, and the like), the separation of target protein is realized by adopting the affinity chromatography technology, and the affinity chromatography has limited treatment capacity and expensive filler medium, thereby limiting the industrial scale-up production of products.

In view of the above situation, the present invention finds purification methods avoiding the use of expensive GST affinity chromatography media for the preparation of MT-III protein pure products, and obtains products with good properties such as and high purity, and simultaneously greatly reduces the purification preparation cost of MT-III protein, so that the large-scale industrial production of MT-III protein is realized.

Disclosure of Invention

The invention aims to provide a preparation method of recombinant human metallothionein III pure products, which is helpful to avoid using expensive GST affinity chromatography column so as to obviously reduce purification preparation cost and obtain high-quality recombinant human metallothionein III pure products.

The task of the invention is to complete the preparation method of recombinant human metallothionein III pure products, which comprises the following steps:

A) preparing a fusion protein solution, namely resuspending the recombinant human metallothionein III fermentation thalli in a buffer solution I, then carrying out ultrasonic crushing, then carrying out centrifugal separation, and collecting a supernatant I to obtain a fusion protein solution containing GST-MT-III;

B) preparing a crude fusion protein GST-MT-III product, carrying out fractional precipitation on the supernatant I obtained in the step A) by using ammonium sulfate, and controlling the technological parameters of the fractional precipitation to obtain the crude fusion protein GST-MT-III product;

C) preparing a supernatant II, namely resuspending the fusion protein GST-MT-III crude product obtained in the step B) in a buffer II to obtain a resuspension solution, adjusting the pH value of the resuspension solution by using a pH regulator, and then performing centrifugal separation to remove insoluble impurity proteins to obtain the supernatant II;

D) preparing enzyme digestion solution, firstly carrying out ultrafiltration on the supernatant II obtained in the step C) by using an ultrafiltration membrane and concentrating to obtain concentrated solution, then adjusting the pH value of the concentrated solution, then adding zinc sulfate and thrombin, and stirring for reaction to obtain the enzyme digestion solution;

E) preparing a crude product liquid of the MT-III protein, heating the enzyme digestion liquid obtained in the step D) to thermally denature and precipitate impurity protein, and performing centrifugal separation to obtain a crude product liquid of the MT-III protein;

F) preparing a semi-finished product solution containing MT-III protein, adjusting the conductivity of the crude product solution of MT-III protein obtained in the step E) until the conductivity of the crude product solution of MT-III protein is equivalent to that of the buffer solution III to obtain the crude product solution of MT-III protein after the conductivity is adjusted, then loading the crude product solution of MT-III protein after the conductivity is adjusted on a hydrophobic chromatographic column which is well balanced in advance, and collecting penetrating liquid to obtain the semi-finished product solution containing MT-III protein;

G) collecting sulfydryl peak eluent, carrying out ultrafiltration desalination on the semi-finished product solution of the MT-III protein obtained in the step F) by using an ultrafiltration membrane, then loading the semi-finished product solution on a well-balanced anion exchange chromatographic column, carrying out gradient elution by using a buffer solution IV and a buffer solution V, and collecting the eluent by separating bottles to obtain sulfydryl peak eluent;

H) and G) preparing a finished product, namely performing ultrafiltration desalting on the sulfydryl peak eluent obtained in the step G), and then freeze-drying the sulfydryl peak eluent into lyophilized powder to obtain a pure recombinant human metallothionein III product.

In specific embodiments of the invention, the volume-to-mass ratio of the buffer solution I and the recombinant human metallothionein III zymocyte in the step A) is 2-5: 1.

In another embodiments of the present invention, the buffer I is or more of phosphate buffer, Tris-HCl buffer and TEA buffer with pH 5-9.

In another specific examples of the present invention, the step B) of fractionating the supernatant I with ammonium sulfate comprises -stage precipitation and two-stage precipitation, and the process parameters for controlling the stage B) refer to the process parameters for controlling the -stage precipitation and the process parameters for controlling the two-stage precipitation.

In another specific embodiments of the invention, the process parameters for controlling -level precipitation include that the temperature of the supernatant I is adjusted, solid ammonium sulfate is slowly added under stirring until the percent saturation of the ammonium sulfate is 20-50%, and the mixture is kept stand to obtain -level precipitate after the ammonium sulfate is completely dissolved, the process parameters for controlling secondary precipitation include that the -level precipitate is centrifugally separated, the supernatant is collected, the temperature of the supernatant is adjusted, the solid ammonium sulfate is slowly added under stirring, the percent saturation of the ammonium sulfate in the supernatant is increased to 60-90%, the mixture is kept stand again after the ammonium sulfate is completely dissolved to obtain secondary precipitate, and the secondary precipitate is centrifugally separated.

In another specific examples of the present invention, the temperature of the supernatant I is adjusted to 20-40 deg.C, the standing is performed at 20-40 deg.C for 60-100min, the temperature of the supernatant is adjusted to 20-40 deg.C, and the standing is performed at 20-40 deg.C for 10-15 h.

In a more specific embodiments of the present invention, the buffer II in step C) is or more of phosphate buffer, Tris-HCl buffer and TEA buffer with pH6-9, and the pH adjustment of the resuspension with a pH adjusting agent is to adjust the pH of the resuspension to 6-9 with 1mol/L aqueous Tris solution.

In a further specific examples, the ultrafiltration of the supernatant II by the ultrafiltration membrane in the step D) is that the supernatant II is subjected to ultrafiltration by an ultrafiltration membrane with molecular weight cut-off of 5-20K, the concentration is that the volume of the ultrafiltered supernatant II is concentrated by 2-5 times, the pH value of the concentrated solution is adjusted to 6-9 by 1mol/L of Tris, the zinc sulfate is added in an amount to make the final concentration of the zinc sulfate in the enzyme digestion solution reach 0.05-0.2mmol/L, the thrombin is an enzyme for specifically shearing a label, the GST is added in an amount of 10000U per 100g of the recombinant human metallothionein III, and the stirring reaction is that the GST is stirred at the temperature of 20-25 ℃ and the speed of 50-100rpm for 14-18 h.

In still more specific embodiments of the present invention, the temperature of the enzyme solution in step E) is 50-80 deg.C, the heating time is 3-10min, the conductivity of the crude MT-III protein solution obtained in step F) is adjusted by using ammonium sulfate as a reagent, the buffer III is or more of phosphate buffer, Tris-HCl buffer and TEA buffer, the pH of the buffer III is 6-9 and contains 1mol/L ammonium sulfate, and the hydrophobic chromatography column is phenyl-agarose gel chromatography column.

In still another specific embodiments of the present invention, the ultrafiltration membrane in step G) is an ultrafiltration membrane with a molecular weight cut-off of 1-3K, the buffer IV is or more of phosphate buffer, Tris-HCl buffer and TEA buffer with a pH value of 6-9, the anion exchange chromatography column is a DEAE sepharose gel chromatography column, the buffer V is a phosphate buffer, Tris-HCl buffer and TEA buffer with a pH value of 6-9 and a final concentration of 0.4mol/L, or more of the buffer, the volume of the thiol peak eluent is 3-5 times that of the DEAE sepharose gel column, the gradient elution is performed by a binary gradient pump to perform gradient mixing elution on the buffer IV and the buffer V in step F), the gradient ratio is changed to that the buffer IV ratio is 100-0% and the buffer V ratio is 0-100% in 2-5 column volume elution time, and the ultrafiltration membrane is used for removing the salt with a desalting molecular weight cut-off of 3K.

The technical scheme provided by the invention has the technical effects that different separation and purification combinations of ammonium sulfate fractional precipitation, hydrophobic chromatographic column chromatography and anion exchange chromatographic column are adopted, so that high-purity recombinant human metallothionein III is obtained, meanwhile, the use of expensive GST affinity chromatography medium is avoided, the preparation cost is obviously reduced, in the enzyme digestion reaction, a large amount of metal ions are required to be chelated in metallothionein molecules with correct folding of a spatial structure, the metal ions are usually zinc, so that the addition of zinc sulfate is very necessary and beneficial to provide quantitative zinc at the initial stage of forming the spatial structure of metallothionein, the addition of zinc sulfate can promote correct folding of the spatial structure of metallothionein and effectively prevent polymerization precipitation of metallothionein fragments, heteroproteins and protein fragments with incorrect spatial conformation, the chelating structure of metallothionein with correct folding of the spatial structure has fixed rigidity due to the fact that a large amount of metal ions are arranged in the space, even if hydrophobic groups of the metallothionein is not easy to interact with hydrophobic medium in a high-salt environment, namely, the protein fragments with incorrect spatial conformation cannot be easily adsorbed on the spatial structure of the recombinant human metallothionein, and the human body can not be better in the recombinant human body, and the human body.

Drawings

FIG. 1 is a diagram of the result of SDS-PAGE electrophoresis detection of the purified recombinant human metallothionein III product obtained by the method of the invention.

Detailed Description

The invention is further illustrated in detail in connection with specific examples which are intended to be illustrative of the invention and are not intended to be limiting.