A label-free OGP self-assembling polypeptide and its preparation method and application

Abstract

The invention discloses a label-free OGP self-assembling polypeptide and a preparation method and application thereof, belonging to the field of biotechnology, and a design sequence SEQ ID NO: 1, which is composed of amino acids, and CTBtriOGP composed of three parts of elements, namely CTB, tri and OGP, can have the osteogenic function of OGP itself. First, a self-assembling OGP gene is obtained by gene synthesis, assembled into an expression vector containing a TAC promoter, transformed into BL21 (DE3) competent bacteria, IPTG induced expression, self-assembling OGP is secreted into the periplasmic cavity of Escherichia coli, CTBtriOGP is extracted from Escherichia coli by osmotic shock method, and CTBtriOGP is obtained by polyethylene glycol centrifugation purification, which not only has the osteogenic function of OGP, but also has stable activity and long storage time, thereby solving the problem of instability of OGP when used alone.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a label-free OGP self-assembling polypeptide and a preparation method and application thereof. Background Art

[0002] Infection and fracture nonunion are common complications after fracture fixation. In recent years, peptide molecules have been used as key drugs for the treatment of bone defects. Osteogenic growth peptide (OGP) is an important peptide growth factor associated with bone regeneration and plays an important role in osteogenesis and hematopoiesis. Osteogenic growth peptide (OGP) and its C-terminal pentapeptide H-Tyr-Gly-Phe-Gly-Gly-OH (OGP10-14) have attracted considerable clinical interest as bone anabolic agents and hematopoietic stimulators. They are present in mammalian serum at micromolar concentrations, increase bone formation and trabecular bone density, and stimulate fracture healing. In culture of osteoblasts and other bone marrow stromal cells from humans and other mammalian species, OGP regulates proliferation, alkaline phosphatase activity, and matrix mineralization through autocrine / paracrine mechanisms. In vivo, it also regulates the expression of type I collagen and basic fibroblast growth factor receptor. In addition, OGP and OGP10-14 enhance hematopoiesis, including stimulating bone marrow transplant engraftment and hematopoietic regeneration after ablative chemotherapy. Apparently, the hematopoietic effects of these peptides are secondary to their effects on the bone marrow stroma.

[0003] Self-assembling peptide nanotechnology has attracted much attention because it can form regular and orderly structures and has diverse functions. Self-assembly is a common phenomenon in nature. In organisms, most proteins function in the form of self-assembly of orderly organizations through non-covalent interactions, such as the formation of cell membranes, protein folding and DNA double helix structures. Inspired by nature, artificially designed protein self-assembly is in full swing. In the past two decades, the field of protein self-assembly has developed rapidly, and a variety of protein assemblies with a wide range of structural and functional properties have been successfully constructed. Studies have found that self-assembling peptides can form aggregates with a certain structure under specific conditions. Such aggregates have the advantages of good biocompatibility and high stability, and show characteristics and advantages different from monomeric peptide molecules. Therefore, they have good application prospects in drug delivery, tissue engineering, antibacterial and other fields.

[0004] Chinese Patent 202010175401.6 discloses the self-assembly ability of the pentamer domain (choleratoxin, CTB) of the bacterial AB5 toxin and a non-natural trimeric peptide, which can produce nanostructures with surface display effects in ordinary Escherichia coli strains. However, the reliance on the histidine tag at the carboxyl terminus will affect the activity of OGP, which requires the carboxyl terminus to be exposed to exert its effect. Summary of the invention

[0005] In view of the deficiencies in the prior art, the object of the present invention is to provide a label-free OGP self-assembling polypeptide and its preparation method and application. Using genetic engineering technology, prokaryotic expression technology or eukaryotic expression technology, label-free purification technology, based on the CTBtri self-assembly platform, the OGP polypeptide is surface displayed, and polyethylene glycol 4000 centrifugal purification and molecular sieve are used for label-free purification, thereby obtaining the self-assembling polypeptide CTBtriOGP with OGP bone-promoting function. This method can avoid the influence of the carboxyl terminal label on the activity of OGP, and can improve the stability and functional activity of the OGP polypeptide. The method of the present invention has broad application prospects in promoting fracture healing and treating bone nonunion.

[0006] The present invention is based on the coding sequence of CTBtri (see patent 202010175401.6), and expresses a polypeptide CTBtriOGP capable of promoting fracture healing by fusing the OGP coding sequence at the carboxyl end. The amino acid sequence of the polypeptide (SEQ ID No: 1) is as follows:

[0007] SEQ ID No: 1:

[0008] MKKIWLALAGLVLAFSASATPQNITDLCAEYHNTQIYTLNDKIFSYTESLAGKREMAIITFKNGAIFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANGGSGRLLLRLEELERRLEELAKFVAAWTLKAAAVDLELAALRRRLEELARGGSG ALKRQGRTLYGFGG.

[0009] The method for preparing the label-free OGP self-assembling polypeptide of the present invention includes the following two methods:

[0010] Method 1 comprises the following steps:

[0011] S1. Construction of vector

[0012] According to the amino acid sequence of CTBtriOGP SEQ ID No: 1, the corresponding coding gene is artificially synthesized using a DNA synthesizer to ensure that the 5' end of CTBtriOGP contains a TAC promoter sequence, a signal peptide sequence and a specific restriction site, and the 3' end contains a terminator sequence and a specific restriction site; the prokaryotic expression vector is cut with the same restriction endonuclease, and then the CTBtriOGP is linked to the multiple cloning site of the prokaryotic expression vector using a DNA ligase;

[0013] S2. Screening of positive transformants and protein expression and purification

[0014] The expression vector containing CTBtriOGP constructed in step S1 was used to transform DH5a or BL21 competent Escherichia coli, and agar plates containing kanamycin resistance were coated to screen positive strains; after the positive transformants were expanded and cultured, they were induced to express in LB medium at low temperature of 4-35°C by IPTG, and CTBTriOGP was expressed in a soluble form and secreted into the periplasmic cavity of the bacteria, and self-assembled into nanospheres with a diameter of about 100 nm; CTBTriOGP was released from the bacteria by osmotic shock method to obtain supernatant, and CTBTriOGP was purified by polyethylene glycol centrifugation method combined with molecular sieve method. The polyethylene glycol molecular weight was 1000-8000, and the concentration was 50%, which was mixed with the supernatant solution obtained by the osmotic shock method in a certain proportion to make the final concentration of PEG reach 10%-25%, and then placed at 4°C overnight, centrifuged at 8000 rpm for 30 minutes, and the supernatant was discarded. An appropriate amount of PBS was added to the precipitate to resuspend it, and the molecular sieve was Superdex 200Increase 10 / 300. GL, the protein peak with the fastest elution is the self-assembled CTBTriOGP, which is collected and dialyzed and freeze-dried to obtain.

[0015] Method 2 comprises the following steps:

[0016] 1) Construction of vector

[0017] According to the amino acid sequence of CTBtriOGP SEQ ID No: 1, the corresponding coding gene is artificially synthesized using a DNA synthesizer to ensure that the 5' end of CTBtriOGP contains a CMV promoter sequence, a signal peptide sequence and a specific restriction site, and the 3' end contains a terminator sequence and a specific restriction site; the eukaryotic expression vector is cut with the same restriction endonuclease, and then the CTBtriOGP is linked to the multiple cloning site of the eukaryotic expression vector using a DNA ligase;

[0018] 2) Screening of positive cell clones and protein expression and purification

[0019] The expression vector containing CTBtriOGP constructed in step 1) was used to transfect CHO cells, and positive cell clones were screened; after the positive cell clones were expanded and cultured, they were expressed in a serum-free suspension medium, and CTBTriOGP was expressed in a soluble form and secreted into the serum-free medium, and self-assembled into nanospheres with a diameter of about 100 nm; after the culture supernatant was concentrated, CTBTriOGP was purified by polyethylene glycol centrifugation combined with molecular sieve method, specifically, polyethylene glycol with a molecular weight of 1000-8000 was prepared to a concentration of 50%, and was mixed with the concentrated supernatant solution in a certain proportion to make the final PEG concentration reach 10%-25%, and was placed at 4°C overnight, centrifuged at 8000 rpm for 30 minutes, and the supernatant was discarded. An appropriate amount of PBS was added to the precipitate to resuspend it, and the molecular sieve was Superdex 200Increase 10 / 300 GL. The protein peak with the fastest peak was the self-assembled CTBTriOGP, which was collected and dialyzed and freeze-dried to obtain it.

[0020] Application of the above-mentioned label-free OGP self-assembling polypeptide in the preparation of a drug for treating bone fractures.

[0021] The present invention has the following beneficial effects:

[0022] (1) The present invention overcomes the shortcoming that the purification of self-assembling polypeptides depends on the carboxyl-terminal tag, and obtains the self-assembling polypeptide CTBtriOGP with OGP activity by a tag-free expression and purification method.

[0023] (2) The osteogenic activity of CTBtriOGP described in the present invention is higher than that of OGP, thus solving the problem of instability and insufficient activity of OGP polypeptide alone.

[0024] The above advantages are conducive to the continuous exertion of OGP activity in the process of fracture healing and have broad application prospects in biopharmaceuticals. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : Electron microscopic identification (upper) and Western blot (lower) results of CTBtriOGP prepared in Example 1-2: Lanes 1 and 2 are the polypeptide prepared in Example 1, and lanes 3 and 4 are the polypeptide prepared in Example 2.

[0026] Figure 2 : The results of CTBtriOGP promoting osteogenic differentiation of bone marrow mesenchymal stem cells: Figure A shows the osteogenic effect of OGP obtained by Nanjing GenScript Biotechnology Co., Ltd. using a conventional solid-phase synthesis method, and Figure B shows the osteogenic effect of the self-assembled CTBtriOGP obtained in Example 1. DETAILED DESCRIPTION

[0027] The following will be combined with the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0028] The present invention is based on the coding sequence of CTBtri (see patent 202010175401.6), and expresses a polypeptide CTBtriOGP capable of promoting fracture healing by fusing the OGP coding sequence at the carboxyl end. The amino acid sequence of the self-assembling polypeptide (SEQ ID No: 1) is as follows:

[0029] SEQ ID No: 1:

[0030] MKKIWLALAGLVLAFSASATPQNITDLCAEYHNTQIYTLNDKIFSYTESLAGKREMAIITFKNGAIFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANGGSGRLLLRLEELERRLEELAKFVAAWTLKAAAVDLELAALRRRLEELARGGSGALKRQGRTLYGFGG

[0031] The method for preparing the self-assembling polypeptide of the present invention includes the following two methods:

[0032] Method 1

[0033] According to the amino acid sequence of CTBtriOGP, the coding gene codon was optimized according to the host cell being Escherichia coli, and the gene sequence of CTBtriOGP was synthesized to ensure that the 5' end of CTBtriOGP contained a TAC promoter sequence, a signal peptide sequence and a specific restriction site, and the 3' end contained a terminator sequence and a specific restriction site; the prokaryotic expression vector was cut with the same restriction endonuclease, and then the CTBtriOGP was linked to the multiple cloning site of the prokaryotic expression vector with DNA ligase, and the expression vector was transformed into DH5a or BL21 Escherichia coli competent cells, and agar plates containing kanamycin resistance were coated to screen positive strains; after the positive transformants were expanded and cultured, they were induced to express in LB medium at a low temperature of 4-35°C by IPTG, and CTBTriOGP was expressed in a soluble form and secreted into the periplasmic cavity of the bacteria, self-assembling into a diameter of about 100 nm nanospheres; CTBTriOGP was released from the bacteria by osmotic shock method, purified by polyethylene glycol centrifugation combined with molecular sieve method, and self-assembling polypeptide with OGP activity was obtained after dialysis and freeze-drying.

[0034] Method 2

[0035] According to the amino acid sequence of CTBtriOGP, the coding gene codons were optimized based on the host cell being Chinese hamster ovary cells, and the gene sequence of CTBtriOGP was synthesized to ensure that the 5' end of CTBtriOGP contained a CMV promoter sequence, a signal peptide sequence and a specific restriction site, and the 3' end contained a terminator sequence and a specific restriction site; the eukaryotic expression vector was cut with the same restriction endonuclease, and then the CTBtriOGP was linked to the multiple cloning site of the eukaryotic expression vector with DNA ligase, and the expression vector was transfected into CHO cells to screen positive cell clones; after the positive cell clones were expanded and cultured, they were expressed in a serum-free suspension medium, and CTBTriOGP was expressed in a soluble form and secreted into the serum-free medium, and self-assembled into nanospheres with a diameter of about 100 nm; after the supernatant of the culture medium was concentrated, CTBTriOGP was purified by a polyethylene glycol centrifugation method combined with a molecular sieve method, and a self-assembling polypeptide with OGP activity was obtained after dialysis and freeze-drying. Example 1

[0036] In this embodiment, E. coli expression was used to prepare CTBtriOGP. The coding gene of CTBtriOGP of the present invention was first synthesized in a biological company and constructed into a prokaryotic secretory expression vector containing a TAC promoter to achieve secretory expression and self-assembly of CTBtriOGP in the periplasm, and PEG4000 was used for label-free purification.

[0037] The specific steps are:

[0038] S1. Vector construction

[0039] According to the amino acid sequence SEQ ID No: 1 of CTBtriOGP of the present invention, the corresponding coding gene is synthesized to ensure that the 5' end contains a signal peptide sequence and a specific restriction site, and the 3' end contains a specific restriction site; the CTBtriOGP gene containing specific restriction sites at both ends and the prokaryotic expression vector (such as Pgex-4T-1) used to express the CTBtriOGP are cut with the same restriction endonuclease, and then the CTBtriOGP is assembled into the multiple cloning site of the secretory prokaryotic expression vector through the specific restriction sites using DNA ligase;

[0040] S2. Screening of positive transformants and protein expression and purification:

[0041] The expression vector containing the CTBtriOGP encoding gene constructed in step S1 is used to transform DH5a or BL21 Escherichia coli competent cells, and the LB culture plate containing kanamycin resistance is coated to screen positive strains; the positive transformed strains are expanded and cultured, and the expression is induced by IPTG at low temperature of 4-35°C in LB medium; the bacteria are collected and washed, and the periplasmic cavity of the bacteria is broken with osmotic shock liquid, and the liquid is centrifuged at low temperature to remove the bacterial precipitation to obtain the supernatant; CTBTriOGP is purified by polyethylene glycol centrifugation combined with molecular sieve method, and the self-assembling polypeptide with OGP activity is obtained after dialysis and freeze-drying. The target protein is identified by western blot with anti-OGP antibody.

[0042] The method optimizes the tag-free expression purification method, does not introduce other amino acids at the OGP carboxyl terminus, and thus produces CTBTriOGP with high OGP activity. The CTBTriOGP can effectively induce osteogenic differentiation of bone marrow mesenchymal stem cells; the tag-free purification method uses polyethylene glycol 4000.

[0043] According to the amino acid sequence described in Sequence 1 (SEQ ID No: 1) of the present invention, the DsbA signal peptide coding sequence was ensured to be contained at the 5' end, and the corresponding gene (sequence SEQ ID No: 2 is as follows) was synthesized and connected to the downstream of the TAC promoter of Pegx-4T-1 using seamless cloning technology. The obtained vector is Pegx-4T-1-CTBtriOGP; the nucleotide sequence of the synthesized gene is as follows:

[0044] . Example 2

[0045] This embodiment uses Chinese hamster ovary cells to express and prepare CTBtriOGP. The coding gene of CTBtriOGP described in the present invention is first synthesized in a biological company and constructed into a eukaryotic secretory expression vector containing a CMV promoter to achieve secretory expression and self-assembly of CTBtriOGP. The serum-free culture medium is recovered and concentrated for label-free purification using PEG4000.

[0046] The specific steps are:

[0047] 1) Vector construction

[0048] According to the amino acid sequence SEQ ID No: 1 of CTBtriOGP of the present invention, the corresponding coding gene is synthesized to ensure that the 5' end contains a signal peptide sequence and a specific restriction site, and the 3' end contains a specific restriction site; the CTBtriOGP gene containing specific restriction sites at both ends and the eukaryotic expression vector (such as pcDNA3.1) used to express the CTBtriOGP are cut with the same restriction endonuclease, and then the CTBtriOGP is assembled into the multiple cloning site of the secretory eukaryotic expression vector through the specific restriction sites using DNA ligase;

[0049] 2) Screening of positive cell clones and protein expression and purification:

[0050] The expression vector containing the CTBtriOGP coding gene constructed in step 1) was used to transfect CHO cells, and positive cell clones were screened using G418; the positive cell clones were expanded and cultured, secreted and expressed in a serum-free medium of CHO cells, and the cells were removed by centrifugation to obtain the cell culture supernatant; CTBTriOGP was purified by polyethylene glycol centrifugation combined with molecular sieve method, and a self-assembling polypeptide with OGP activity was obtained after dialysis and freeze-drying. The target protein was identified by western blot using anti-OGP antibody.

[0051] The method optimizes the tag-free expression purification method, does not introduce other amino acids at the OGP carboxyl terminus, and thus produces CTBTriOGP with high OGP activity. The CTBTriOGP can effectively induce osteogenic differentiation of bone marrow mesenchymal stem cells, and the tag-free purification method uses polyethylene glycol 4000.

[0052] Test on promoting osteogenic differentiation of bone marrow mesenchymal stem cells: Bone marrow mesenchymal stem cells were seeded in 6-well plates at 105 cells / well, and the culture medium containing OGP polypeptide and CTBtriOGP polypeptide was replaced the next day. After 7 days of osteogenic induction, the culture medium was discarded, and the cells were gently washed once with PBS. 1 mL of 4% paraformaldehyde solution was added to each well for fixation for 10 minutes, and the fixative was discarded; after fixation, the cells were washed 3 times with PBS, 3-5 minutes each time, and attention was paid to being gentle; the required volume of BCIP / NBT staining working solution was prepared according to the instructions of the ALP staining kit; 1 mL of BCIP / NBT staining working solution was added to each well to ensure that each well could fully cover the sample; incubated at room temperature and in the dark for 5-30 minutes until the color was developed to the expected depth; the BCIP / NBT staining working solution was removed, and the color development reaction was terminated by washing with distilled water 1-2 times, and the residual liquid was removed to complete the color development. The osteogenic induction effect was judged according to the depth of the color. The darker the color, the better the effect. The test results are as follows: Figure 2As shown, Figure A shows the osteogenic effect of OGP obtained by Nanjing GenScript Biotechnology Co., Ltd. using a conventional solid phase synthesis method, and Figure B shows the osteogenic effect of the self-assembled CTBtriOGP obtained in Example 1. The results show that the osteogenic effect of the self-assembled CTBtriOGP provided by the present invention is significantly better than the osteogenic effect of OGP obtained by the conventional solid phase synthesis method.

[0053] Although the embodiments of the present application have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present application, and that the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a tag-free OGP self-assembling polypeptide, wherein the polypeptide is based on the coding sequence of CTBtri and is expressed by fusing the OGP coding sequence at the carboxyl end. The amino acid sequence of the tag-free OGP self-assembling polypeptide CTBtriOGP is MKKIWLALAGLVLAFSASATPQNITDLCAEYHNTQIYTLNDKIFSYTESLAGKREMAIITFKNGAIFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANGGSGRLLLRLEELERRLEELAKFVAAWTLKAAAVDLELAALRRRLEELARGGSG ALKRQGRTLYGFGG, characterized in that: The following steps are involved: S1. Construction of vector According to the amino acid sequence of CTBtriOGP, the corresponding coding gene was artificially synthesized using a DNA synthesizer to ensure that the 5' end of CTBtriOGP contained a TAC promoter sequence, a signal peptide sequence and a specific restriction site, and the 3' end contained a terminator sequence and a specific restriction site; the prokaryotic expression vector Pgex-4T-1 was cut with the same restriction endonuclease, and then the CTBtriOGP was connected to the multiple cloning site of the prokaryotic expression vector using DNA ligase; S2. Screening of positive transformants and protein expression and purification The expression vector containing CTBtriOGP constructed in step S1 is used to transform DH5a or BL21 competent Escherichia coli, and agar plates containing kanamycin resistance are coated to screen positive strains; after the positive transformants are expanded and cultured, they are induced to express in LB medium at low temperature of 4-35°C by IPTG, and CTBTriOGP is expressed in a soluble form and secreted into the periplasmic cavity of the bacteria, and self-assembled into nanospheres with a diameter of 100 nm; CTBTriOGP is released from the bacteria by an osmotic shock method, and CTBTriOGP is purified by a polyethylene glycol centrifugation method combined with a molecular sieve method, and dialyzed and freeze-dried to obtain.

2. A method for preparing a tag-free OGP self-assembling polypeptide, wherein the polypeptide is based on the coding sequence of CTBtri and is expressed by fusing the OGP coding sequence at the carboxyl end. The amino acid sequence of the tag-free OGP self-assembling polypeptide CTBtriOGP is MKKIWLALAGLVLAFSASATPQNITDLCAEYHNTQIYTLNDKIFSYTESLAGKREMAIITFKNGAIFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANGGSGRLLLRLEELERRLEELAKFVAAWTLKAAAVDLELAALRRRLEELARGGSG ALKRQGRTLYGFGG, characterized in that: The following steps are involved: 1) Construction of vector According to the amino acid sequence of CTBtriOGP, the corresponding coding gene was artificially synthesized using a DNA synthesizer to ensure that the 5' end of CTBtriOGP contained a CMV promoter sequence, a signal peptide sequence and a specific restriction site, and the 3' end contained a terminator sequence and a specific restriction site; the eukaryotic expression vector pcDNA3.1 was cut with the same restriction endonuclease, and then the CTBtriOGP was connected to the multiple cloning site of the eukaryotic expression vector using DNA ligase; 2) Screening of positive cell clones and protein expression and purification The expression vector containing CTBtriOGP constructed in step 1) is used to transfect CHO cells, and positive cell clones are screened; after the positive cell clones are expanded and cultured, they are expressed in a serum-free suspension medium, and CTBTriOGP is expressed in a soluble form and secreted into the serum-free medium, and self-assembles into nanospheres with a diameter of 100 nm; after the supernatant of the culture medium is concentrated, CTBTriOGP is purified by a polyethylene glycol centrifugation method combined with a molecular sieve method, and dialyzed and freeze-dried to obtain.

3. Use of a label-free OGP self-assembling polypeptide prepared according to the preparation method according to claim 1 or 2 in the preparation of a drug for treating bone fractures.

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