CN118240054A - Method for purifying Sox2 protein - Google Patents

Abstract

The invention discloses a method for purifying Sox2 protein. The method comprises the step of purifying Sox2 protein using salting out. The method provided by the invention has the advantages of low cost, simple operation, good preparation of buffer liquid, high yield and high purity of the purified Sox2 protein, and has wide application prospect.

Description

Method for purifying Sox2 protein

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for purifying Sox2 protein.

Background

CN104962531a discloses a method for extracting lactoperoxidase by ultrafiltration assisted ammonium sulfate salting-out, which uses bovine whey as raw material, adopts ammonium sulfate salting-out method to separate lactoperoxidase in bovine whey, specifically comprises adding ammonium sulfate with a certain saturation into whey, precipitating lactoperoxidase for a period of time, centrifuging to completely precipitate lactoperoxidase, collecting precipitate to obtain lactoperoxidase, redissolving the precipitate, removing small impurity protein by ultrafiltration membrane, further purifying lactoperoxidase, desalting by dialysis bag, and freeze-drying to obtain the lactoperoxidase product with high activity which can be used as natural food additive.

CN103740689B discloses a method for purifying chymotrypsin by affinity chromatography and sectional elution, which comprises the following steps: (1) multiple crystallization; (2) activation; (3) salting out; (4) ultrafiltration; (5) pyrogen treatment; (6) dialysis; (7) affinity chromatography; (8) lyophilizing to prepare chymotrypsin. The invention continuously improves the production process of chymotrypsin, particularly repeatedly carries out experimental study on various process parameters, continuously optimizes, finally establishes a set of more scientific large-scale production process, obtains chymotrypsin with the potency of up to 1500-1800 iu/mg through affinity chromatography purification, and meets the specification of Chinese pharmacopoeia in each index.

CN102911926a relates to a method for producing high-purity trypsin by adopting an affinity chromatography method, which comprises the steps of raw material acquisition for production, raw material crushing, protein extraction, fractional salting-out, zymogen precipitation, crude product enzymolysis, affinity chromatography separation and purification, ultrafiltration concentration and sterilization, and vacuum freeze drying to obtain a finished product. Compared with the prior art, the invention has the advantages of producing trypsin by using an affinity chromatography, is beneficial to the simplification of the process and the quality control, reduces the production cost, improves the product quality, obtains the product with high purity (more than 3000 units/mg) with higher yield, and greatly improves the market competitiveness of the product.

CN106754841a relates to an affinity chromatography preparation method of high-activity trypsin, which takes bovine pancreas or porcine pancreas as raw material, comprising the following steps: crushing raw materials; coarse extraction of pancreatic protein; two-stage salting out of ammonium sulfate; performing enzymolysis and activation on the crude product; taking GE Health gel medium Sepharose CL 4B as a framework, taking (2, 6 dihydroxy) heptyl (6 amino) acetyl para-aminophenylguanidine as a ligand, and preparing an affinity chromatography filler for affinity chromatography under alkaline conditions for 24 hours; concentrating and sterilizing by ultrafiltration; vacuum freeze drying to obtain the final product. The method avoids complex zymogen precipitation steps in the traditional process, greatly simplifies the working procedures, can obtain the high-purity and high-activity product with higher yield, has stable process and controllable quality, greatly reduces the production cost and improves the market competitiveness of the product.

CN109307771a relates to a method for quantitatively detecting the content of an intermediate in the production process of recombinant human alpha interferon by affinity chromatography, which comprises the following steps: 1) Constructing engineering bacteria for expressing recombinant human alpha interferon; 2) Fermenting engineering bacteria, and separating and purifying target protein expressed in the form of soluble protein or inclusion body. The protein purification comprises crude purification and fine purification, wherein ammonium sulfate salting-out precipitation is utilized to obtain a crude protein product, the crude protein product is purified by a DEAE anion exchange column chromatography to obtain an intermediate, and the content of recombinant human alpha interferon in the intermediate is quantitatively detected by a monoclonal antibody affinity chromatography column. The method is suitable for quantitative detection of recombinant human alpha interferon and intermediate of recombinant integrated interferon production process, and has the advantages of simple, quick and accurate operation, and can improve the cognition degree and control degree of people on the production process according to the obtained interferon concentration data, so that reliable, quick, direct and easy evaluation of the key links of interferon production is possible, and the aim of accurate quality control is fulfilled.

The transcription factor Sox2 is one of the Sox region Y related HMG (High Mobility Group) protein family members. The SOX protein family plays an important role in early mammalian organ development, in particular the transcription factor SOX2.Sox2 is one of the necessary factors for regulating mammalian embryo development. Sox2 is a transcription factor, and has been studied more frequently for its use in regulatory mechanisms, antibodies, and medicine, and has been very scarce in terms of resources for purification process study after its expression. As chromatographic techniques mature, the development of protein purification was strongly driven. Overall, the purification of Sox2 proteins is focused on column chromatography technology.

In the prior art (Zhang Qi et al, "clone expression and purification of human Sox2 gene". In the university of Huazhong, university of academy of sciences: nature science 42.1 (2008): 4.), human Sox2 protein was amplified and expressed by E.coli BL23 (DE 3), and the obtained inclusion body was cleaved by a buffer system containing 8M Urea. Then purifying and renaturation are carried out on a chromatographic column, the medium used by the column chromatography is Ni Sepharose 6Fast Flow Instruction (GE), and the protein yield after renaturation is 0.7mg/g wet bacterial weight.

The method for preparing the humanized Sox2 protein has the following defects: (1) The 8M Urea system is not easy to prepare, and cannot realize the amplification process in a GMP workshop with strict temperature control. (2) the yield is only 0.7mg/g wet weight, and the yield is low. In the process of washing inclusion bodies, 1M Urea is used for removing impurities, and a large amount of target protein is lost in the step, so that the yield is affected. (3) The production cost is high, and the 8M Urea system and the GE medium both increase the production cost.

Disclosure of Invention

The invention aims to overcome the defects that in the prior art, when human Sox2 protein is purified, the cost is high, a buffer solution system used for purification is difficult to prepare, an amplification process in a GMP workshop with strict temperature control cannot be realized, and the yield of Sox2 protein is low, and provides a method for purifying Sox2 protein. The method provided by the invention has the advantages of low cost, simple operation, good preparation of buffer liquid, high yield and high purity of the purified Sox2 protein, and has wide application prospect.

The invention solves the technical problems through the following technical proposal.

The first aspect of the present invention provides a method for purifying Sox2 protein, the method comprising the step of purifying Sox2 protein using salting out.

In some embodiments of the invention, the salting-out is fractional salting-out.

In some embodiments of the invention, the salt used for salting out is ammonium sulfate.

In some preferred embodiments of the invention, the fractional salting is two-stage salting.

In some preferred embodiments of the invention, the concentration of ammonium sulfate in the two-stage salting-out is 20-25% and 25-30%, respectively.

In some examples of the invention, the concentration of ammonium sulfate in the two-stage salting-out is 25 and 30%, respectively.

In some embodiments of the invention, the salting out is performed at 0-4 ℃.

In some embodiments of the invention, the method further comprises the step of affinity chromatography.

In some preferred embodiments of the invention, the affinity chromatography is nickel affinity chromatography.

In some preferred embodiments of the invention, the nickel affinity chromatography uses NW Rose Ni FF media.

In some embodiments of the invention, the affinity chromatography comprises the steps of:

(1) Front balance; the equilibration buffer used comprises urea, PB, naCl and/or imidazole;

(2) Loading a sample;

(3) Post-balancing; the equilibration buffer used comprises urea, PB, naCl and/or imidazole;

(4) Washing; the wash buffer used contained urea, PB, naCl, imidazole and/or Triton X-114;

(5) Eluting; the elution buffer used comprises urea, PB, naCl and/or imidazole.

In some preferred embodiments of the invention, the equilibration buffer used in (1) and (3) comprises 5-6M urea, 5-20mM PB, 50-300mM NaCl and 2-5mM imidazole;

(4) Wherein the washing is three-step washing; and/or the number of the groups of groups,

(5) The elution buffers used in (1) are 5-6M urea, 5-20mM PB, 200-500mM NaCl and 400-500mM imidazole;

In some preferred embodiments of the invention, the wash buffer used in the three-step wash described in (2) comprises 5-6M urea, 5-20mM PB, 800-1000mM NaCl, and 2-10mM imidazole, respectively; 5-6M urea, 5-20mM PB, 50-300mM NaCl, 2-10mM imidazole and 0.1% -0.2% Triton X-114; and 5-6M urea, 5-20mM PB, 50-300mM NaCl, and 10-20mM imidazole.

In some embodiments of the invention, the equilibration buffer used in (1) is 5-10 column volumes; the equilibration buffer used in (3) is 3-5 column volumes; (4) The washing buffer solution used in the three-step washing is 5-10 column volumes, 5-10 column volumes and 3-5 column volumes; and/or (5) using elution buffer of 3-5 column volumes.

In some examples of the invention, the affinity chromatography comprises the steps of:

(1) Front balance: the equilibration buffer used contained 6M urea, 20mM PB, 287mM NaCl and 5mM imidazole; 10 column volumes;

(2) Loading a sample;

(3) Post-equilibration: the equilibration buffer used contained 6M urea, 20mM PB, 287mM NaCl and 5mM imidazole; 5 column volumes;

(4) Washing:

(a) High salt washing: the wash buffer 1 used contained 6M urea, 20mM PB, 1000mM NaCl and 5mM imidazole; 10 column volumes;

(b) Triton wash: washing buffer 2 used contained 6M urea, 20mM PB, 287mM NaCl, 5mM imidazole and 0.2% Triton X-114;10 column volumes;

(c) And (3) final washing: the wash buffer 3 used contained 6M urea, 20mM PB, 200mM NaCl and 20mM imidazole; 5 column volumes;

(5) Eluting: the elution buffers used were 6M urea, 20mM PB, 500mM NaCl and 500mM imidazole.

In some embodiments of the invention, the method meets one or more of the following conditions:

the pH of the balance buffer is 7-8.2;

the pH of the washing buffer is 7-8.2;

The pH of the elution buffer is 7-8.2.

In a second aspect the invention provides a method as described in the first aspect, wherein the Sox2 protein has an amino acid sequence as shown in SEQ ID No. 1 or has at least 90% identity thereto.

On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.

The reagents and materials used in the present invention are commercially available.

The invention has the positive progress effects that:

The method provided by the invention has the advantages of low cost, simple operation, good preparation of buffer liquid, high yield of purified Sox2 protein (up to 6.98mg/g protein/thallus) and high purity (up to 98.3%), and has wide application prospect.

Drawings

FIG. 1 is a diagram showing the result of SDS-PAGE electrophoresis of purified human Sox2 protein by affinity chromatography. Wherein E1, E2, E3 respectively represent the first, second and third tubes (and so on) collected in separate tubes, respectively, eluting the product.

FIG. 2 is a diagram showing SDS-PAGE electrophoresis of protein precipitate after salting out with ammonium sulfate. Wherein, 20-25% sediment represents that the concentration of ammonium sulfate in the first-stage salting-out is 20%, and the concentration of ammonium sulfate in the second-stage salting-out is 25%; the 25-30% sediment represents that the concentration of ammonium sulfate in the first-stage salting-out is 25% and the concentration of ammonium sulfate in the second-stage salting-out is 30%; the 30-40% sediment represents that the concentration of ammonium sulfate in the first-stage salting-out is 30% and the concentration of ammonium sulfate in the second-stage salting-out is 40%; the 40-50% sediment represents that the concentration of ammonium sulfate in the first-stage salting-out is 40% and the concentration of ammonium sulfate in the second-stage salting-out is 50%;50-60% precipitation means that the concentration of ammonium sulfate in the first stage salting-out is 50% and the concentration of ammonium sulfate in the second stage salting-out is 60%.

FIG. 3 is a diagram showing the result of SDS-PAGE electrophoresis of the purified human Sox2 protein using ammonium sulfate salting out and purification process 1. Wherein E1, E2, E3 respectively represent the first, second and third tubes (and so on) collected in separate tubes, respectively, eluting the product.

FIGS. 4A and 4B are graphs showing the results of SDS-PAGE electrophoresis of the human Sox2 protein purified by the ammonium sulfate salting-out and purifying process 2. Wherein 200-1, 200-2 respectively represent the elution products of a first tube and a second tube (and the like) collected by the branch tube when the concentration of imidazole in the eluent is 200 mM; 300-1, 300-2 respectively represent the eluted products of the first tube and the second tube collected by the branch tube (and so on) when the imidazole concentration in the eluent is 300 mM; 500-1, 500-2 respectively represent the eluted product from the first and second tubes (and so on) collected by the separate tube when the imidazole concentration in the eluate was 500 mM.

FIGS. 5A and 5B are graphs showing the results of SDS-PAGE electrophoresis of the purified human Sox2 protein using ammonium sulfate salting out and purification process 3. Wherein 50-1 and 50-2 respectively represent the eluted products of the first tube and the second tube (and so on) collected by the branch tube when the NaCl concentration in the eluent is 50 mM; 100-1, 100-2 respectively represent the eluted products of the first tube and the second tube (and so on) collected by the branch tube when the NaCl concentration in the eluent is 100 mM; 200-1, 200-2 respectively represent the eluted products of the first tube and the second tube (and so on) collected by the branch tube when the NaCl concentration in the eluent is 200 mM; 500-1 and 500-2 represent the eluted products from the first and second tubes (and so on) collected by the separate tubes, respectively, when the NaCl concentration in the eluate was 500 mM.

FIG. 6 is a diagram showing the result of SDS-PAGE electrophoresis of the purified human Sox2 protein using ammonium sulfate salting out and purification process 4. Wherein E1, E2, E3 respectively represent the first, second and third tubes (and so on) collected in separate tubes, respectively, eluting the product.

FIG. 7 is a diagram showing the result of SDS-PAGE electrophoresis of the purified human Sox2 protein using ammonium sulfate salting out and purification process 5. Wherein E1, E2, E3 and E4 represent the first, second, third and fourth tube eluate products collected in separate tubes, respectively.

FIG. 8 is a diagram showing the result of SDS-PAGE electrophoresis of the purified human Sox2 protein using ammonium sulfate salting out and purification process 6. Wherein E1, E2, E3 respectively represent the first, second and third tubes (and so on) collected in separate tubes, respectively, eluting the product.

FIG. 9 is a diagram showing the result of SDS-PAGE electrophoresis of the purified human Sox2 protein using ammonium sulfate salting out and purification process 7. Wherein E1, E2, E3 respectively represent the first, second and third tubes (and so on) collected in separate tubes, respectively, eluting the product.

FIG. 10 is a diagram showing the results of HPLC detection for purifying human Sox2 protein by using ammonium sulfate salting out and purifying process 7.

FIG. 11 is a non-reducing SDS-PAGE of purified human Sox2 protein using ammonium sulfate salting-out purification process 7.

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

The sequence of the human Sox2 protein used in the present invention is shown in table 1.

TABLE 1 sequence of human Sox2 protein

Example 1

In this example, the human Sox2 protein was purified by affinity chromatography.

E.coli expressing recombinant human Sox2 protein was resuspended in equilibration buffer (6M Urea+20mM PB+287mM NaCl+5mM IM,pH 8.2) at a ratio of E.coli: equilibration buffer=1:10 (mass to volume ratio), sonicated 2 times at 900.+ -. 50bar, centrifuged 2 times at 10000rpm (4 ℃,30 min/time), and the supernatant cell lysate was collected and stirred overnight to serve as an affinity chromatography sample. Purification of Sox2 protein was performed using NW Rose Ni FF affinity chromatography media (su sodium micro technologies inc.) packed chromatography column.

The column was equilibrated 10 Column Volumes (CV) with equilibration buffer (6M Urea+20mM PB+287mM NaCl+5mM IM,pH 8.2) for pre-equilibration. The cell lysate obtained by the ultrasonic disruption was diluted three times (the turbidity of the sample was about 86 NTU) and then was applied. At the end of loading, 5CV was equilibrated with equilibration buffer. After washing with high salt, tritonX-114, W1 and W2, elution was performed by adding elution buffer (6M Urea+20mM PB+500mM IM-500mM NaCl) and collecting the eluted product in separate tubes.

The buffers and conditions for the purification steps are shown in Table 2.

TABLE 2 purification step by affinity chromatography

When the cell lysates obtained by disrupting 14g of cells and 28g of cells were purified, the yields were 22.91mg/g (protein/cell) and 17.5mg/g, respectively. The eluted products collected from the separation tube were examined by a reduction SDS-PAGE method, and as shown in FIG. 1, there were a plurality of hetero protein bands in addition to the Sox2 protein of interest (i.e., the coarsest band in FIG. 1), indicating that the purity of the product obtained by purification by affinity chromatography alone was poor, and it was necessary to perform crude purification of the cell lysate by ammonium sulfate salting out method and then further chromatography.

Example 2

The Sox2 protein obtained by affinity chromatography purification has poor purity, so that ammonium sulfate is added to carry out crude purity on the cell lysate. E.coli expressing recombinant human Sox2 protein was resuspended in buffer (6M Urea+10mM PBS+150mM NaCl+20mM IM,pH 7.6) at a ratio of E.coli: buffer=1:10 (mass to volume ratio), sonicated 2 times at 900.+ -.50 bar, centrifuged 2 times at 10000rpm (4 ℃,30 min/time), and the supernatant was collected as cell lysate for ammonium sulfate precipitation. Adding ammonium sulfate (the concentration in the mixed solution is 20%, 25%, 30%, 40% and 50%, respectively) into the cell lysate for primary salting out, stirring at 4 ℃ for at least 6h, centrifuging at 10000rpm and 4 ℃ for 60min, and discarding the precipitate; continuing to add ammonium sulfate (the concentration in the mixed solution is 25%, 30%, 40%, 50% and 60%) into the supernatant for secondary salting out, stirring at 4 ℃ for at least 6h, centrifuging at 10000rpm and 4 ℃ for 60min, discarding the supernatant, and reserving protein sediment.

As shown in fig. 2, at 40% ammonium sulfate concentration (secondary chromatography), the target protein was almost completely precipitated; the purity of Sox2 protein is higher when the gradient is 25-30% (namely the gradient of primary ammonium sulfate is 25% and the gradient of secondary ammonium sulfate is 30%), and the yield percentage of the strain is about 25-35%. The primary ammonium sulfate gradient was determined to be 25% and the secondary ammonium sulfate gradient was determined to be 30% and this partial precipitation was subsequently used for subsequent purification optimization.

Example 3

The precipitate obtained after crude purification by ammonium sulfate salting-out with 25-30% ammonium sulfate in example 2 was resuspended in buffer=1:50 (mass to volume ratio) in equilibration buffer (6M Urea+20mM PB+287mM NaCl+5mM IM), stirred slowly for more than 10h to re-lyse, followed by subsequent column purification. The ammonium sulfate salting-out method comprises the following steps of: adding 25% ammonium sulfate into the cell lysate, stirring at 4deg.C for at least 6h, centrifuging at 10000rpm at 4deg.C for 60min, and discarding the precipitate; continuing to add ammonium sulfate to the supernatant to a concentration of 30%, stirring at 4 ℃ for at least 6h, centrifuging at 10000rpm and 4 ℃ for 60min, discarding the supernatant, and retaining the precipitate.

Purification process 1:

subsequent purification was performed using NW Rose Ni FF affinity chromatography media (su sodium micro technologies inc.) packed chromatography column. The purification steps and conditions are shown in table 3.

TABLE 3 purification procedure 1

The eluted products were collected by sorting, and the result of detection by reducing SDS-PAGE was shown in FIG. 3, and the purity of the obtained Sox2 protein was not high, and the yield of Sox2 protein was 6.15mg/g (protein/cell).

Purification process 2:

And filling the chromatographic column with NW Rose Ni FF affinity chromatography medium for subsequent purification. The procedure and conditions are shown in Table 4, and elution is performed by setting an imidazole concentration gradient.

Table 4 purification process 2 steps

The eluted products were collected in separate tubes. The result of detection by reducing SDS-PAGE of the eluted products is shown in FIGS. 4A and 4B, and the purity of the obtained Sox2 protein is not high. The yields of Elutation 1, elutation 2 and Elutation 3 were 0.50, 1.75 and 4.69mg/g (protein/cell).

Purification process 3:

Subsequent purification was performed by packing the column with Unigel-50SP ion exchange chromatography media (Soy micro technologies Co., ltd.). The procedure and conditions are shown in Table 5, and elution is performed by setting a NaCl concentration gradient.

Table 5 purification procedure 3

Step (a)	Step is abbreviated as	Buffer and conditions
			-	Column conditions	Unigel-50SP,50mL/CV,15mL/min, room temperature, pH 8.2
1	Front balance	6M Urea+20mM PB；10cv
			2	Loading sample	10g
3	Post-balancing	6M Urea+20mM PB；5cv
			4	Elutation 1 Elution	6M Urea+20mM PB+50mM NaCl
5	Elutation 2 Elution	6M Urea+20mM PB+100mM NaCl
			6	Elutation 3 Elution	6M Urea+20mM PB+200mM NaCl
7	Elutation 4 Elution	6M Urea+20mM PB+500mM NaCl

The eluted products were collected by separating tubes, and the eluted products were not high in purity as shown in FIGS. 5A and 5B in the detection results of reduced SDS-PAGE. The yields of Elutation 1, elutation 2, elutation 3 and Elutation 4 were 0.87, 5.42, 2.94 and 0.04mg/g (protein/cell), respectively.

Purification process 4:

purifying sequentially by Unigel-50SP ion exchange chromatography medium and NW Rose Ni FF affinity chromatography medium. The procedure and conditions are shown in Table 6.

Table 6 purification procedure of Process 4

The eluted product was obtained after affinity chromatography, and the result of SDS-PAGE detection was shown in FIG. 6, wherein the purity of Sox2 protein was not high, and the yield was 4.64mg/g (protein/cell).

Purification process 5:

the purification was performed sequentially using Unigel-80Q ion exchange chromatography media (Soviet micro technologies Co., ltd.) and NW Rose Ni FF affinity chromatography media. The procedure and conditions are shown in Table 7.

TABLE 7 purification Process 5 steps

The eluted product was obtained after affinity chromatography, and the result was shown in FIG. 7, in which the purity of the obtained Sox2 protein was not high and the yield of Sox2 protein was 6.81mg/g (protein/cell).

The technical schemes of the purification processes 4 and 5 are that ion exchange chromatography is carried out first and then affinity chromatography is carried out. From the detection result of the obtained product, when ion exchange chromatography and affinity chromatography are performed first, the whole ion column is in an ineffective impurity removal state. In the purification process 3, the Sox2 protein is purified by adopting Unigel-50SP ion exchange chromatography medium, and the Sox2 protein with high purity is not obtained when NaCl with different concentration gradients is used for step elution during elution. In the purification process 2, sox2 protein purification is performed by using NW Rose Ni FF affinity chromatography medium, and high purity Sox2 protein is not obtained even when imidazole with different concentration gradients is used for stepwise elution. The purity of the protein obtained by the purification process is far less than ideal purity, so that the washing and impurity removal process is optimized mainly from one-step affinity.

Purification process 6:

Purification was performed using NW Rose Ni FF affinity chromatography medium. The procedure and conditions are shown in Table 8.

Table 8 purification procedure 6

As a result of detection by reducing SDS-PAGE, the yield of Sox2 protein was 0.75mg/g (protein/cell) as shown in FIG. 8.

Purification process 7:

Purification was performed using NW Rose Ni FF affinity chromatography medium. The procedure and conditions are shown in Table 9.

Table 9 purification procedure of process 7

200MM NaCl is added into the buffer used for the W1 washing in the purification process 6, so that the stripping of the target protein is promoted, and a large amount of protein is lost in the W1 washing step. Thus, in purification process 7, the imidazole content in the W1 wash is reduced. The eluted products were collected by a separate tube and detected by reducing SDS-PAGE, and the results are shown in FIG. 9. The yield of Sox2 protein was 6.98mg/g (protein/cell). The eluted product of the branched tube was subjected to sample matching, and the HPLC detection result is shown in FIG. 10 and Table 10, and the purity of the purified product of Sox2 protein was 98.3%.

Table 10HPLC peak detection table

Peak number	Retention time	Area of	Height of (1)	Marking	Concentration of	Area percent
							1	7.071	95151	13674	M	1.513	1.513
2	7.285	3201349	177882	V M	50.916	50.916
							3	7.914	2882804	157377	V M	45.850	45.850
4	8.421	108215	5205	V M	1.721	1.721
							Totals to		6287519	354138		100.000	100.000

Two large peaks appear in the figure because the proteins in the elion exist mostly as dimers, monomers and small amounts of multimers. Reference can be made to a non-reducing SDS-PAGE (without addition of DTT) detection of the purified Sox2 protein product as shown in FIG. 11.

The two-stage salting-out method of ammonium sulfate and the purification process 7 are combined to obtain the optimized Sox2 protein purification method, the purity of the obtained purified Sox2 protein is 98.3%, and the yield is 6.98mg/g (protein/thallus).

Claims (10)

1. A method of purifying Sox2 protein, characterized in that the method comprises a step of purifying Sox2 protein using salting out.

2. The method of claim 1, wherein the salting-out is fractional salting-out; and/or the salt used for salting out is ammonium sulfate.

3. The method of claim 2, wherein the staged salting-out is a two-stage salting-out;

Preferably, the concentration of ammonium sulfate in the two-stage salting-out is 20-25% and 25-30% respectively;

More preferably, the concentration of ammonium sulfate in the two-stage salting-out is 25 and 30%, respectively.

4. A method according to any one of claims 1 to 3, wherein the salting out is performed at 0 to 4 ℃.

5. The method of any one of claims 1-4, further comprising the step of affinity chromatography;

Preferably, the affinity chromatography is nickel affinity chromatography, preferably using NW Rose Ni FF medium.

6. The method of claim 5, wherein the affinity chromatography comprises the steps of:

(1) Front balance; the equilibration buffer used comprises urea, PB, naCl and/or imidazole;

(2) Loading a sample;

(3) Post-balancing; the equilibration buffer used comprises urea, PB, naCl and/or imidazole;

(4) Washing; the wash buffer used contained urea, PB, naCl, imidazole and/or Triton X-114;

(5) Eluting; the elution buffer used comprises urea, PB, naCl and/or imidazole.

7. The method of claim 6, wherein the equilibration buffer used in (1) and (3) comprises 5-6M urea, 5-20mM PB, 50-300mM NaCl, and 2-5mM imidazole;

(4) Wherein the washing is three-step washing; and/or the number of the groups of groups,

(5) The elution buffers used in (1) are 5-6M urea, 5-20mM PB, 200-500mM NaCl and 400-500mM imidazole;

Preferably, the three-step wash described in (2) uses wash buffers comprising 5-6M urea, 5-20mM PB, 800-1000mM NaCl and 2-10mM imidazole, respectively; 5-6M urea, 5-20mMPB, 50-300mM NaCl, 2-10mM imidazole and 0.1% -0.2% Triton X-114; and 5-6M urea, 5-20mM PB, 50-300mM NaCl, and 10-20mM imidazole.

8. The method of claim 7, wherein the equilibration buffer used in (1) is 5-10 column volumes; the equilibration buffer used in (3) is 3-5 column volumes; (4) The washing buffer solution used in the three-step washing is 5-10 column volumes, 5-10 column volumes and 3-5 column volumes; and/or (5) using elution buffer of 3-5 column volumes.

9. The method of any one of claims 6-8, wherein the method satisfies one or more of the following conditions:

the pH of the balance buffer is 7-8.2;

the pH of the washing buffer is 7-8.2;

The pH of the elution buffer is 7-8.2.

10. A method according to any one of claims 1 to 9, wherein the Sox2 protein has an amino acid sequence as shown in SEQ ID No.1 or at least 90% identity thereto.