KR101905261B1 - Method for producing recombinant protein of silkworm storage protein 1 using fructose - Google Patents

Abstract

The present invention relates to a method for producing a recombinant protein of silkworm storage protein 1 (SP1) using fructose, and more particularly, to a method for producing recombinant SP1 protein produced by cell culture by size exclusion chromatography chromatography, the addition of a certain amount of fructose to the elution buffer suppresses the aggregation phenomenon of the recombinant SP1, and even after concentration through subsequent ultrafiltration, A recombinant SP1 protein produced by the silkworm storage protein 1, a recombinant SP1 protein produced by the silkworm SP1 protein and its inhibition of apoptosis, a cell culture Culture media for the production of biopharmaceuticals, use as anti-oxidation or functional (cosmetic) materials will be.

Description

TECHNICAL FIELD The present invention relates to a method for producing a recombinant protein of a silkworm silkworm protein protein using a fructose,

The present invention relates to a method for producing a recombinant protein of silkworm storage protein 1 (SP1) using fructose, and more particularly, to a method for producing recombinant SP1 protein produced by cell culture by size exclusion chromatography chromatography, the addition of a certain amount of fructose to the elution buffer suppresses the aggregation phenomenon of the recombinant SP1, and even after concentration through subsequent ultrafiltration, A recombinant SP1 protein produced by the silkworm storage protein 1, a recombinant SP1 protein produced by the silkworm SP1 protein and its inhibition of apoptosis, a cell culture Culture media for the production of biopharmaceuticals, use as anti-oxidation or functional (cosmetic) materials will be.

In the biopharmaceutical industry, the apoptosis that occurs during the cultivation of animal cells through biological incubators is becoming a big problem. During cell culture in a biological incubator, the cells are placed in environments where cell death is induced, such as hypoxia, lack of nutrients, and shear stress. The decrease in cell viability due to apoptosis results in a decrease in the production of recombinant protein. Furthermore, apoptosis in cell culture increases the risk of creating unwanted byproducts and requires further purification to remove these by-products.

Therefore, in order to increase the productivity of biopharmaceuticals and to eliminate the necessity of further processing in the production process, it is necessary to develop a cell death inhibiting substance and utilize it as a cell culture medium additive.

Meanwhile, the domestic cosmetics market is steadily growing, and the market for functional cosmetics is growing rapidly. With the development of science, the life span of human beings has been getting longer, and due to factors such as low fertility, it has rapidly entered into an aging society. In the cosmetics industry, there is a long-term need to respond, and interest in functional cosmetics is expected to grow. Therefore, it is required to develop functional cosmetics having higher efficacy in accordance with consumer needs. Specifically, humans experience aging phenomena in which physiological functions are degraded with time. The degree of aging varies according to each individual, and skin is the biggest indicator that can judge the extent of the individual's biological aging. Since the skin is always exposed to the external environment, a lot of skin changes due to aging can be attributed to the influence of external factors. Among the external factors, ultraviolet rays and active oxygen generated through respiration are regarded as the most important cause of skin aging. Active oxygen leads to skin cell and tissue damage and destroys anti-oxidation defense systems.

Therefore, it is important to prevent and aging skin aging by aging caused by active oxygen, which has a great influence on skin aging at present, and it is time to develop new cosmetic materials having excellent anti-oxidative action in this regard.

The inventors of the present invention have confirmed through previous studies that silkworm storage protein 1 (SP1) exerts cytotoxic and anti-oxidative effects on mammalian cells including humans have. That is, when a cell capable of producing the SP1 protein is used to produce an apoptosis-resistant cell, the cell is resistant to various stresses generated during the culture, and a cell line for the production of an antibody or the like can be developed using the cell . The antioxidant function of SP1 also suggests possibility of functional cosmetic material which inhibits skin aging.

In order to increase the utilization of SP1 in the biotechnology industry, it is necessary to mass-produce SP1 as a recombinant protein. In this case, the recombinant SP1 protein produced may be used as an additive to a cell culture medium for production of biopharmaceuticals, and it is highly possible to develop anti-aging cosmetic materials utilizing anti-oxidation.

In this regard, the present inventors produced a SP1 expression vector and inserted it into Escherichia coli to produce transformed cells, thereby producing SP1 as a recombinant protein and purifying it.

Since the expression vector containing the SP1 gene has a histidine tag attached to the C terminus, the affinity column capable of binding to the histidine tag after cell disruption of the water-soluble SP1 in the cultured cells was used to express 1 SP1 can be separated from cellular impurities. The SP1 can then be purified using size exclusion chromatography and concentrated through ultrafiltration to increase the concentration of purified SP1.

However, when the concentration of SP1 is increased in the purification process of SP1, aggregation phenomenon occurs, and especially in the case of superfine filtration, such coagulation phenomenon occurs severely, so that it is necessary to solve this problem in order to produce high concentration of recombinant SP1 .

Kim, E. J., W. J. Rhee, and T. H. Park (2001) Isolation and Characterization of an Apoptosis-Inhibiting Component from the Hemolymph of Bombyx mori. Biochem. Biophys. Res. Commun. 285; 224-228.

DISCLOSURE OF THE INVENTION The present invention has been made to solve the above-mentioned conventional needs. As a result of intensive studies, the present inventors have found that when fructose is added to the elution buffer in the elution process of size exclusion chromatography, It was confirmed that the aggregation phenomenon was effectively suppressed (even after ultrafine filtration), and the present invention was accomplished. The purified recombinant SP1 retained cell death inhibition and anti - oxidative effects when added to the culture medium.

That is, the present invention provides a novel method of mass-producing SP1 at a high concentration in the form of a recombinant protein without causing coagulation at the time of purification using fructose and a variety of uses based on the biological function of the recombinant SP1 protein thus produced It is a technical task.

In order to accomplish the above object, the present invention provides a method for producing a recombinant protein of silkworm storage protein 1 (SP1), wherein the produced recombinant SP1 is subjected to Size Exclusion Chromatography (or molecular exclusion chromatography) , Wherein the fructose is added to an elution buffer used for the elution (or elution) of the size exclusion chromatography. The fructose-containing silkworm Lt; RTI ID = 0.0 > 1 < / RTI >

The above-mentioned storage protein 1 (SP1) is a kind of insect hemocyanin protein isolated from silkworm hemolymph, which is a female-specific protein of silkworm and contains a large amount (10% or more) of methionine.

Specifically, the storage protein 1 (SP1) may have an amino acid sequence represented by the following sequence listing.

[SP1 amino acid sequence] Among them, SP1 used in the present invention corresponds to Thr 16 amino acids.

For example, Korean Patent No. 10-0530506, or Kim, EJ, WJ Rhee, and TH Park (2001), which is a prior study of the present inventor, discloses a method for isolating and purifying SP1 from a body fluid, Isolation and Characterization of an Apoptosis-Inhibiting Component from the Hemolymph of Bombyx mori . Biochem. Biophys . Res . Commun . 285: 224-228. "And Rhee, WJ, EH Lee, JH Park, JE Lee, and TH Park (2007) Inhibition of HeLa cell apoptosis by storage-protein 2. Biotechnol . Prog . 23: 1441-1446. &Quot;

Specifically, the present invention provides

a) introducing the SP1 expression vector into Escherichia coli to prepare transformed cells;

b) culturing the transformed cells to produce recombinant SP1;

c) disrupting the cells from which recombinant SP1 has been produced, followed by affinity chromatography to primarily isolate SP1 from cellular impurities;

d) purifying SP1 by performing size exclusion chromatography using an elution buffer to which fructose is added to the separated product; And

e) concentrating the purified product by ultrafiltration.

A method for producing a recombinant protein of a storage protein 1 of silkworm by using fructose is provided.

The SP1 expression vector (for example, the SP1 gene inserted in the pET23a vector) may have a histidine tag attached thereto. In this way, the soluble SP1 in the cultured cells can be firstly disrupted from the cell impurities by affinity column capable of binding to Histidine tag after cell disruption. On the other hand, the storage protein 1 (SP1) gene inserted into the vector may be a gene accession number NM_001113276 and the E. coli may be a BL21 (DE3) strain.

In the cell disruption and affinity chromatography in the step c), the pellet obtained in the step b) is suspended in a lysis buffer, sonicated and centrifuged to separate into supernatant and pellet, Lt; RTI ID = 0.0 > SP1 < / RTI > by affinity chromatography. In this case, there is a soluble form of SP1 in the separated supernatant, and an insoluble form of SP1 is present in the separated pellet.

The amount of fructose to be added to the elution buffer (elution solvent) when performing size exclusion chromatography in step d) is added in an amount sufficient to prevent aggregation of the recombinant SP1, for example, to a concentration of about 2 M . As the elution buffer, any of the types commonly used in the art for size exclusion chromatography for protein purification can be employed without any particular limitation. On the other hand, impurities such as imidazole are removed through this step, and pure recombinant SP1 protein can be purified.

The step e) of the step e) is a step of concentrating the supernatant by ultrafiltration (ultrafiltration or ultrafiltration), and in the case of the present invention, when the fructose is added, the recombinant SP1 There is a distinct advantage that does not aggregate.

According to another aspect of the present invention there is provided a recombinant SP1 protein produced / purified according to the method as described above.

The recombinant SP1 protein according to the present invention is useful for the treatment of cell death (apoptosis or apoptosis), particularly apoptosis induced by mammals, including humans, in particular by intrinsic apoptosis pathway, such as Staurosporine. Lt; RTI ID = 0.0 > (STS). ≪ / RTI > Accordingly, the present invention can significantly increase the cell viability of cells during cell culture, thereby greatly increasing the productivity of biopharmaceuticals and the like. Meanwhile, SP1, which is a target of mass production of the present invention, does not share any sequence homology with existing apoptosis inhibitors such as Bcl-2, 30K protein or IAP (Inhibitor of apoptosis protein) Can be treated as an inhibitor.

In addition, the present inventors have experimentally confirmed the effect of SP1 on human cell apoptosis, specifically, the anti-apoptosis effect of SP1 on Hela cells.

Therefore, the recombinant SP1 protein according to the present invention can be suitably used for the production of cell cultures for the production of additives for cell culture medium for the production of biopharmaceuticals (for example, proteins), or biopharmaceuticals (for example, proteins). Here, the biologic drug may be a monoclonal antibody as a therapeutic protein.

In addition, the recombinant SP1 protein according to the present invention has an anti-oxidizing effect and an active oxygen inhibiting effect in addition to the apoptosis inhibiting effect. As a result, the recombinant SP1 protein according to the present invention can be suitably used not only as general cosmetics but also as a material for functional cosmetics such as anti-aging.

The recombinant SP1 protein production method according to the present invention can effectively inhibit the aggregation of SP1 generated during the purification process. Especially, the aggregation suppression effect is remarkably exhibited even after concentration through ultrafiltration, There is an advantage to be able to produce.

In addition, when the purified recombinant SP1 protein was added to the culture medium, it still retained apoptosis inhibition and anti-oxidation (and reactive oxygen inhibition) effects.

Accordingly, the present invention is a method for maximizing productivity by solving the problems occurring in the process of mass production of SP1 having such functions in the field where inhibition of cell death is required or anti-oxidation material is required.

Figure 1 shows SDS-PAGE, Western blot and I-TASSER results of the recombinant SP1 protein.
Fig. 2 shows the yield analysis results of the recombinant SP1 protein in each purification step.
Figure 3 is a graph showing anti-apoptosis and anti-oxidative effects of recombinant SP1 protein.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. It should be understood, however, that these examples are for illustrative purposes only and are not intended to limit the scope of the invention in any way.

Example

(1) microbial culture and SP1  production

Colonies of strain BL21 (DE3) -pET23a (+) SP1 streaked on a plate (w / Amp.) Were collected and seeded (OD600 0.6-0.8) in 5 mL of LB broth (w / , And cultured (OD600 0.6-0.8) by inoculation on 1 L LB broth.

IPTG (0.5 mM) was added thereto, followed by incubation at 30 ° C for 12 hours and induction.

The pellet was suspended in a lysis buffer, sonicated and centrifuged to separate the supernatant and the pellet.

At this time, soluble form (soluble form) of SP1 was present in the supernatant, insoluble form of SP1 was present in the pellet, and filtration was performed to purify SP1 from the supernatant.

(2) SP1  Purification and concentration

Affinity chromatography was performed on the supernatant by filtration using a His-Trap column.

The His-tag binds to the recombinant SP1 protein, followed by washing and elution to isolate only the SP1 protein.

Size exclusion chromatography was performed to remove the cytotoxic substance imidazole which was mixed in the elution process.

Using the principle that the order in which they come out depends on the size, only the SP1 protein that precedes the smaller imidazole was taken. At this time, a 2 M solution of fructose was used to prevent aggregation of SP1 protein.

The SP1 protein solution thus separated was concentrated by ultrafiltration and quantified.

Experimental Example

(1) Recombination SP1  Protein SDS - PAGE , Western blot  And I- TASSER  result

SDS-PAGE loaded with the soluble form (lane # 1), refolded form (lane # 2) and insoluble form (lane # 3) of recombinant SP1 (rSP1) The results are shown in Figure 1A. At this time, 2, 1 and 0.5 μg of each sample were quantified using the BSA standard.

In addition, western blotting of the water-soluble form (lane # 1), insoluble form (lane # 2) and purified rSP1 (lane # 3) using an anti- .

Through these, the recombinant SP1 produced according to the present invention could be detected and quantified.

Fig. 1C shows the visualization result of the structure of rSP1 through I-TASSER.

(2) Recombination SP1  Yield analysis results for each purification of protein

In order to analyze the yield in each purification process, the products of the process were subjected to band quantification by SDS-PAGE, and the results are shown in FIG.

As shown in FIG. 2, it can be confirmed that the recombinant SP1 protein can be finally obtained at a high yield and concentration according to the present invention.

(3) Recombination SP1  The anti- Apoptosis  And anti-oxidative effect

To analyze the anti-apoptosis effect of rSP1, rSP1 was incubated with cells (e.g., HeLa cells) and then treated with 1 μM of staurosporine (STS), and after 0, 12, Cell viability was measured by trypan blue exclusion assay, and the results are shown in FIG. 3A.

In order to analyze the anti-oxidative stress effect of rSP1, rSP1 was incubated with the cells and treated with 0.6 M H ₂ O ₂ for 0, 12, and 18 hours, The cell viability was measured through analysis, and the results are shown in FIG. 3B.

As shown in FIGS. 3A and 3B, it can be confirmed that the purified recombinant SP1 protein using fructose according to the present invention still exerts excellent anti-apoptosis and anti-oxidative effects.

<110> INCHEON UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> METHOD FOR PRODUCING RECOMBINANT PROTEIN OF SILKWORM STORAGR          PROTEIN 1 USING FRUCTOSE <130> P16-0090 <140> 10-2016-0110261 <141> 2016-08-29 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 747 <212> PRT <213> Bombyx mori <400> 1 Met Arg Val Leu Val Leu Leu Ala Cys Leu Ala Ala Ala Ser Ala Thr   1 5 10 15 Ala Ile Ser Gly Gly Tyr Gly Thr Met Val Phe Thr Lys Glu Pro Met              20 25 30 Val Asn Leu Asp Met Lys Met Lys Glu Leu Cys Ile Met Lys Leu Leu          35 40 45 Asp His Ile Leu Gln Pro Thr Met Phe Glu Asp Ile Lys Glu Ile Ala      50 55 60 Lys Glu Tyr Asn Ile Glu Lys Ser Cys Asp Lys Tyr Met Asn Val Asp  65 70 75 80 Val Val Lys Gln Phe Met Glu Met Tyr Lys Met Gly Met Leu Pro Arg                  85 90 95 Gly Glu Thr Phe Val His Thr Asn Glu Leu Gln Met Glu Glu Ala Val             100 105 110 Lys Val Phe Arg Val Leu Tyr Tyr Ala Lys Asp Phe Asp Val Phe Met         115 120 125 Arg Thr Ala Cys Trp Met Arg Glu Arg Ile Asn Gly Gly Met Phe Val     130 135 140 Tyr Ala Phe Thr Ala Ala Cys Phe His Arg Thr Asp Cys Lys Gly Leu 145 150 155 160 Tyr Leu Pro Ala Pro Tyr Glu Ile Tyr Pro Tyr Phe Phe Val Asp Ser                 165 170 175 His Val Ile Ser Lys Ala Phe Met Met Lys Met Thr Lys Ala Ala Lys             180 185 190 Asp Pro Val Leu Trp Lys Tyr Tyr Gly Ile Thr Val Thr Asp Asp Asn         195 200 205 Leu Val Val Ile Asp Trp Arg Lys Gly Val Arg Arg Ser Leu Ser Gln     210 215 220 Asn Asp Val Met Ser Tyr Phe Met Glu Asp Val Asp Leu Asn Thr Tyr 225 230 235 240 Met Tyr Tyr Leu His Met Asn Tyr Pro Phe Trp Met Thr Asp Asp Ala                 245 250 255 Tyr Gly Ile Asn Lys Glu Arg Arg Gly Glu Ile Met Met Tyr Ala Asn             260 265 270 Gln Gln Leu Leu Ala Arg Met Met Leu Glu Arg Leu Ser His Lys Met         275 280 285 Cys Asp Val Lys Pro Met Met Trp Asn Glu Pro Leu Glu Thr Gly Tyr     290 295 300 Trp Pro Lys Ile Arg Leu Pro Ser Gly Asp Glu Met Pro Val Arg Gln 305 310 315 320 Asn Asn Met Val Val Ala Thr Lys Asp Asn Leu Lys Met Lys Gln Met                 325 330 335 Met Asp Asp Val Glu Met Met Ile Arg Glu Gly Ile Leu Thr Gly Lys             340 345 350 Ile Glu Arg Arg Asp Gly Thr Val Ile Ser Leu Lys Lys Ser Glu Asp         355 360 365 Ile Glu Asn Leu Ala Arg Leu Val Leu Gly Gly Leu Glu Ile Val Gly     370 375 380 Asp Asp Ala Lys Val Ile His Leu Thr Asn Leu Met Lys Lys Met Leu 385 390 395 400 Ser Tyr Gly Gln Tyr Asn Met Asp Lys Tyr Thr Tyr Val Pro Thr Ser                 405 410 415 Leu Asp Met Tyr Thr Thr Cys Leu Arg Asp Pro Val Phe Trp Met Ile             420 425 430 Met Lys Arg Val Cys Asn Ile Phe Thr Val Phe Lys Asn Met Leu Pro         435 440 445 Lys Tyr Thr Arg Glu Gln Phe Ser Phe Pro Gly Val Lys Val Glu Lys     450 455 460 Ile Thr Thr Asp Glu Leu Val Thr Phe Val Asp Glu Tyr Asp Met Asp 465 470 475 480 Ile Ser Asn Ala Met Tyr Leu Asp Ala Thr Glu Met Gln Asn Lys Thr                 485 490 495 Ser Asp Met Thr Phe Met Ala Arg Met Arg Arg Leu Asn His His Pro             500 505 510 Phe Gln Val Ser Ile Asp Val Met Ser Asp Lys Thr Val Asp Ala Val         515 520 525 Val Arg Ile Phe Leu Gly Pro Lys Tyr Asp Cys Met Gly Arg Leu Met     530 535 540 Ser Val Asn Asp Lys Arg Leu Asp Met Phe Glu Leu Asp Ser Phe Met 545 550 555 560 Tyr Lys Leu Val Asn Gly Lys Asn Thr Ile Val Arg Ser Ser Ser Met Asp                 565 570 575 Met Gln Gly Phe Ile Pro Glu Tyr Leu Ser Thr Arg Arg Val Met Glu             580 585 590 Ser Glu Met Met Pro Ser Gly Asp Gly Gln Thr Met Val Lys Asp Trp         595 600 605 Trp Cys Lys Ser Arg Asn Gly Phe Pro Gln Arg Leu Met Leu Pro Leu     610 615 620 Gly Thr Ile Gly Gly Leu Glu Met Gln Met Tyr Val Ile Val Ser Pro 625 630 635 640 Val Arg Thr Gly Met Leu Leu Pro Thr Leu Asp Met Thr Met Met Lys                 645 650 655 Asp Arg Cys Ala Cys Arg Trp Ser Ser Cys Ile Ser Thr Met Pro Leu             660 665 670 Gly Tyr Pro Phe Asp Arg Pro Ile Asp Met Ala Ser Phe Phe Thr Ser         675 680 685 Asn Met Lys Phe Ala Asp Val Met Ile Tyr Arg Lys Asp Leu Gly Met     690 695 700 Ser Asn Thr Ser Lys Thr Val Asp Thr Ser Glu Met Val Met Met Lys 705 710 715 720 Asp Asp Leu Thr Tyr Leu Asp Ser Asp Met Leu Val Lys Arg Thr Tyr                 725 730 735 Lys Asp Val Met Met Met Ser Ser Met Met Asn             740 745

Claims (19)

delete a) preparing a transformed cell by introducing a pET23a vector into which a histidine tag is attached and a SP1 gene is inserted into E. coli BL21 (DE3);
b) culturing the transformed cells to produce recombinant SP1;
c) disrupting the cells from which the recombinant SP1 has been produced, then subjecting the SP1 present in the supernatant to affinity chromatography using a His-Trap column to firstly separate SP1 from the cell impurity;
d) removing the imidazole and purifying SP1 by performing size exclusion chromatography using an elution buffer to which 2 M of fructose is added to the separated product; And
e) concentrating the purified product by ultrafiltration,
Wherein the fructose inhibits aggregation of SP1 and the SP1 protein retains anti-apoptosis and anti-oxidative effects after concentration.
A method for producing recombinant proteins of stored protein 1 of silkworm using fructose.
delete delete delete 3. The method of claim 2,
The step c)
After the cells cultured in step b) were centrifuged, the pellet was suspended in a lysis buffer, sonicated, centrifuged, and separated into supernatant and pellet.
Characterized in that SP1 present in the supernatant is separated by affinity chromatography.
A method for producing recombinant proteins of stored protein 1 of silkworm using fructose.
The method according to claim 6,
Characterized in that the supernatant has a soluble form of SP1 and the pellet has an insoluble form of SP1.
A method for producing recombinant proteins of stored protein 1 of silkworm using fructose.


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