Mutant streptococcus protein G and application thereof in purification of horse serum antibody F (ab ') 2 fragment
By designing a mutant streptococcal protein G that specifically targets the F(ab′)2 fragment in horse serum, the problems of low purification efficiency, low purity, and environmental pollution in existing technologies have been solved, achieving efficient and environmentally friendly purification of the F(ab′)2 fragment.
Patent Information
- Application Number
- CN202511247190.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-02
- Publication Date
- 2025-11-14
AI Technical Summary
Existing technologies are inefficient, yield-low, involve multiple steps, and produce low purity when purifying F(ab′)2 fragments from horse serum. Furthermore, traditional methods are harmful to the environment. Protein G affinity chromatography cannot effectively separate F(ab′)2 and Fc fragments.
A mutant streptococcal protein G was designed to specifically target the F(ab′)2 fragment without binding to the Fc fragment. This protein was then coupled with agarose to prepare an adsorbent material, which was used as a chromatography column packing material to achieve efficient purification of the F(ab′)2 fragment from horse serum.
This method enables rapid and efficient purification of the F(ab′)2 fragment, breaking through the purity and yield bottlenecks of traditional methods. It is suitable for the automation needs of large-scale production and reduces the risk of environmental pollution.
Smart Images
Figure CN120943909A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and in particular to a mutant streptococcal protein G and its application in the purification of equine serum antibody F(ab′)2 fragment. Background Technology
[0002] As a large immunizing animal, horses' serum can be efficiently used to prepare high-titer polyclonal antibodies against pathogens such as rabies virus and tetanus toxin. However, the Fc fragment of equine IgG contains strong heterologous antigenic epitopes, which can trigger severe immune rejection reactions when directly applied to humans. To reduce immunogenicity, current processes require pepsin digestion to convert full-length IgG into F(ab′)2 and Fc fragments, followed by purification techniques to remove the Fc fragment and purify the F(ab′)2 fragment.
[0003] Currently, the industry mainly uses ammonium sulfate precipitation to purify F(ab′)2 fragments, but this technology has significant drawbacks: ① some antibodies are denatured and inactivated, resulting in a lower yield; ② the product purity is low, and further purification is usually required; ③ high-salt waste liquid is harmful to the environment and increases environmental pressure.
[0004] Affinity chromatography can also achieve purification by utilizing the natural binding properties of Protein G in the packing material. Protein G primarily targets the Fc fragment of antibodies, but it also has an affinity for F(ab′)2, making it unsuitable for separating F(ab′)2 and the Fc fragment. Therefore, it is necessary to improve the separation efficiency of Protein G. Summary of the Invention
[0005] Based on the technical problems existing in the background technology, the present invention proposes a mutant streptococcal protein G and its application in the purification of horse serum antibody F(ab′)2 fragment. The mutant protein G of the present invention can rapidly and efficiently purify F(ab′)2 fragment from horse serum by specifically targeting the F(ab′)2 fragment without binding to the Fc fragment, thus solving the industry pain points of low efficiency, low yield, multiple steps and low product purity of existing processes.
[0006] This invention proposes a mutant streptococcal protein G, the amino acid sequence of which is shown below:
[0007] ①
[0008] TTYKLVINGKTLKGETTTKAVDAETAX1X2AFKQYANDNGVDGVWTYDDATKTFTVTE; where X1 is independently selected from one of K, R, and H; and X2 is independently selected from one of E and D.
[0009] ② An amino acid sequence that has more than 80% homology with the amino acid sequence shown in ①, and still has affinity for the F(ab′)2 fragment but no affinity for the Fc fragment.
[0010] The aforementioned homology can be 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc.
[0011] Preferably, the mutant streptococcal protein G has an affinity for the F(ab′)2 fragment but no affinity for the Fc fragment.
[0012] Preferably, the F(ab′)2 fragment is the F(ab′)2 fragment of horse serum antibody and the Fc fragment is the Fc fragment of horse serum antibody.
[0013] Preferably, the amino acid sequence of the mutant protein G is shown in SEQ ID NO: 1-6.
[0014] The present invention also proposes a polynucleotide that encodes the above-mentioned mutant streptococcal protein G.
[0015] The aforementioned polynucleotides refer to chain-like compounds formed by the polymerization of nucleotides; the aforementioned polynucleotides include DNA or RNA.
[0016] The present invention also proposes an expression vector containing the above-mentioned polynucleotides.
[0017] The present invention also proposes a host cell containing the above-mentioned expression vector or the above-mentioned polynucleotides integrated into the host cell genome.
[0018] Preferably, the host cell is a cell capable of expressing exogenous proteins.
[0019] Preferably, the host cell is a bacterium, yeast, insect cell, or mammalian cell.
[0020] The present invention also proposes a fusion protein comprising: the above-mentioned mutant streptococcal protein G, and having a 6×His tag attached to the C-terminus of the amino acid sequence of mutant streptococcal protein G.
[0021] The amino acid sequence of the above 6×His tag is HHHHHH.
[0022] The present invention also proposes an adsorbent material, wherein the above-mentioned mutant streptococcal protein G or the above-mentioned fusion protein is immobilized on a carrier to obtain the adsorbent material.
[0023] The aforementioned carriers can be: inorganic carriers (such as glass beads, silica gel, etc.), synthetic polymers (such as cross-linked polyvinyl alcohol, cross-linked polyacrylate, cross-linked polyacrylamide, and cross-linked polystyrene, etc.), crystalline cellulose (such as cross-linked dextran and other organic carriers composed of polysaccharides, organic-organic carriers composed of these, organic-inorganic carriers, organic-inorganic carriers, etc., composite carriers), agarose, etc. These carriers can be in any form, such as beads, fibers, membranes, etc.
[0024] The method of immobilization on the carrier is not limited to the above-mentioned method; for example, reactive functional groups (such as epoxy groups) can be introduced into the surface of the carrier and the mutant protein G or the fusion protein can be used as ligands to form covalent bonds on the carrier through reaction.
[0025] Preferably, the carrier is agarose.
[0026] Preferably, the agarose is pre-activated with epoxy.
[0027] Epoxy pre-activated agarose is available commercially.
[0028] This invention also proposes the application of the above-mentioned mutant streptococcal protein G, the above-mentioned fusion protein, and the above-mentioned adsorption material in the separation and purification of F(ab′)2 fragments and their derivatives, and as packing materials for chromatography columns.
[0029] Preferably, the F(ab′)2 fragment is the F(ab′)2 fragment of horse serum antibody.
[0030] The present invention also proposes a method for separating and purifying the F(ab′)2 fragment, comprising the following steps: using the above-mentioned adsorbent material as the chromatographic column packing material, performing column chromatography on the sample containing the F(ab′)2 fragment to obtain the F(ab′)2 fragment.
[0031] Preferably, the F(ab′)2 fragment is the F(ab′)2 fragment of horse serum antibody.
[0032] Preferably, the eluent for column chromatography is an aqueous solution of acetic acid.
[0033] Preferably, the pH of the acetic acid aqueous solution is 2.8-3.2.
[0034] This invention targets the structures of streptococcal protein G and the F(ab′)2 and Fc fragments of horse serum antibody. Based on structural biology techniques, sequence modification was performed. Mutations were designed at positions 297-352 of the amino acid sequence of wild-type streptococcal protein G. Multiple mutant sequences were designed, and after screening, six mutant amino acid sequences were obtained. These six amino acid sequences were all obtained by mutating amino acids at positions 323-324. The mutation design eliminates the affinity of streptococcal protein G for the Fc fragment of horse serum antibody, but it has good affinity for the F(ab′)2 fragment of horse serum antibody and has specific targeting ability.
[0035] The resulting mutant streptococcal protein G can rapidly and efficiently purify the F(ab′)2 fragment from horse serum by specifically targeting the F(ab′)2 fragment without binding to the Fc fragment, thus solving the industry pain points of low efficiency, low yield, multiple steps, and low product purity of existing processes.
[0036] Experiments have shown that the adsorbent material prepared by coupling mutant streptococcal protein G with agarose can specifically capture the F(ab′)2 fragment from horse serum without binding the Fc fragment. This method enables efficient one-step capture of the target F(ab′)2 fragment, overcoming the purity and yield bottlenecks of traditional precipitation methods and meeting the automation requirements of large-scale production. Attached Figure Description
[0037] Figure 1 The images show the protein purification results of Protein G-M1. In Figure A, the results of Protein G-M1 protein purification by nickel column are shown; and in Figure B, the results of Protein G-M1 protein identification by SDS-PAGE are shown.
[0038] Figure 2 A represents the SPR identification results of the binding ability of wild-type Protein G-WT and mutant Protein G-M1 to the Fc fragment of equine serum antibody, where PG-WT is wild-type Protein G and PG-M1 is Protein G-M1.
[0039] Figure 2 BC represents the kinetic constants for the binding ability of wild-type Protein G-WT and mutant Protein G-M1 to the F(ab′)2 fragment of equine serum antibody. In this figure, B represents wild-type Protein G and C represents Protein G-M1.
[0040] Figure 3The images show the SDS-PAGE results of proteins purified from horse serum antibody F(ab′)2 and Fc fragments using a Protein G-M1 affinity column. In the images, A shows the purification results of the affinity column on an AKTA protein purifier, B shows the SDS-PAGE results, 1 shows the elution product, 2 shows the substance obtained from column flow-through, and 3 shows the mixture before column purification. Detailed Implementation
[0041] The technical solution of the present invention will be described in detail below through specific embodiments. However, it should be clearly stated that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention.
[0042] Example 1: Preparation of mutant protein G
[0043] The inventors designed a mutation at positions 297-352 of the G amino acid sequence of wild-type Streptococcus protein.
[0044] Since there are currently no literature reports on the complex structures of horse serum antibody Fc fragments, F(ab′)2 fragments, and streptococcal protein G, and given the high homology of Fc and F(ab′)2 fragments across different species, the inventors consulted the Protein Structure Database (PDB) and referenced the complex structures of streptococcal protein G and human serum antibody Fc fragments from 1FCC (PDB number), and the complex structures of streptococcal protein G and human and mouse serum antibody Fab fragments from 6OC7 and 1IGC (PDB numbers). They analyzed the interacting amino acid residues in the complex structures and mutated these residues to disrupt the original interactions. They discovered that mutating amino acids at positions 323-324 of wild-type streptococcal protein G to specific amino acids reversed the acid-base relationship of these positions, thus reversing the charge of key interacting residues. This changed the charge attraction between Protein G and the Fc fragment at this site into charge repulsion, thereby obtaining a complex structure without affecting the interaction of protein G and Fc fragments. While maintaining the affinity of Protein G for the F(ab′)2 fragment of equine serum antibody, mutant sequences were designed to reduce or even completely eliminate the affinity of Protein G for the Fc fragment of equine serum antibody. A total of 6 mutant Streptococcus protein G gene sequences were designed, and their amino acid sequences are shown in SEQ ID NO: 1-6.
[0045] The mutant streptococcal protein G shown in SEQ ID NO: 1 is named Protein G-M1, and its DNA sequence is shown in SEQ ID NO: 7.
[0046] The amino acid sequences shown in SEQ ID NO: 2-6 correspond to the DNA sequences shown in SEQ ID NO: 8-12.
[0047] The protein G-M1 gene sequence was synthesized, and primers were designed to insert the wild-type streptococcal protein G and protein G-M1 gene sequences into the Escherichia coli expression vector pET30a, respectively. A 6×His tag was added to the C-terminus of each amino acid sequence to obtain the recombinant vectors, which were then sequenced and stored.
[0048] Each recombinant vector was transformed into competent Escherichia coli Rosetta, and E. coli were cultured on a large scale. The expression of the target protein was induced by 1 mM IPTG. After overnight induction at 16°C, the bacterial cells were collected and lysed by sonication. The supernatant of each group was collected separately, and then purified by nickel ion affinity chromatography to obtain the fusion protein of each group.
[0049] The binding affinity of the fusion proteins in each group to the Fc and F(ab′)2 fragments of equine serum antibody was initially detected by ELISA. It was found that the mutant protein Protein G-M1 had affinity for the F(ab′)2 fragment of equine serum antibody, but not for the Fc fragment of equine serum antibody.
[0050] The fusion proteins were taken and added to SDS-loading buffer, heated in a metal bath at 100°C for 5 minutes, and then run on gradient gels. Coomassie Brilliant Blue was used for staining and destaining. Testing revealed that the purity of the fusion proteins was above 95%, meeting the requirements for affinity identification and subsequent preparation of agarose filler.
[0051] Typical images such as Figure 1 As shown. Figure 1 The images show the protein purification results of Protein G-M1. In Figure A, the results of Protein G-M1 protein purification by nickel column are shown; and in Figure B, the results of Protein G-M1 protein identification by SDS-PAGE are shown.
[0052] Depend on Figure 1 It can be seen that: Figure 1 Lanes 1, 2, 3, 4, and 5 in B correspond to elution volumes of 50 mL, 55 mL, 60 mL, 65 mL, and 70 mL, respectively. The results indicate that the purified Protein G-M1 protein has a purity of over 95%, which meets the requirements for affinity identification and subsequent preparation of agarose packing material.
[0053] Example 2
[0054] Affinity determination of protein G to equine serum antibody Fc and F(ab′)2 fragments
[0055] Preparation of Fc and F(ab′)2 fragments of horse serum antibody
[0056] Horse serum was collected, and the full-length IgG antibody in the horse serum was purified using a standard recombinant Protein G affinity chromatography column. The full-length IgG antibody was then digested with pepsin to obtain F(ab′)2 and Fc fragments, which were then separated by molecular sieve gel filtration chromatography to obtain high-purity F(ab′)2 and Fc fragments.
[0057] SPR analysis of the binding affinity of protein G to the Fc fragment of equine serum antibody
[0058] The binding of wild-type Protein G (Protein G-WT) and mutant Protein G (Protein G-M1) to the Fc fragment was detected using a Biacore 8K instrument. The specific steps included: coating the same amount of Protein G-WT or each mutant Protein G onto a CM5 chip, then passing each chip through a 10 μg / mL Fc fragment, and detecting the binding signal. Results are as follows: Figure 2 As shown in Figure A.
[0059] Figure 2 A represents the SPR identification results of the binding ability of wild-type Protein G-WT and mutant Protein G-M1 to the Fc fragment of equine serum antibody, where PG-WT is wild-type Protein G and PG-M1 is Protein G-M1.
[0060] Depend on Figure 2 As can be seen from A: when the coating is wild-type Protein G, a response value of approximately 170 Ru is generated, indicating that the horse serum antibody Fc fragment binds to wild-type Protein G; when the coating is Protein G-M1, there is no response value, indicating that the horse serum antibody Fc fragment does not bind to Protein G-M1, suggesting that the mutation eliminates the binding ability of Protein G to the Fc fragment.
[0061] SPR analysis of the kinetic constant of protein G against equine serum antibody F(ab′)2 fragment.
[0062] Using a Biacore 8K instrument, equal amounts of wild-type Protein G (Protein G-WT) and mutant Protein G (Protein G-M1) were coated onto a CM5 chip. A multi-cycle kinetic constant detection method was employed, and binding signals were detected using F(ab′)2 fragment solutions at different dilutions (initial concentration 400 nM, serially diluted twofold). Results are as follows: Figure 2 As shown in BC.
[0063] Figure 2 BC represents the kinetic constants for the binding ability of wild-type Protein G-WT and mutant Protein G-M1 to the F(ab′)2 fragment of equine serum antibody. In this figure, B represents wild-type Protein G and C represents Protein G-M1.
[0064] Depend on Figure 2 BC shows that the equilibrium dissociation constant K of wild-type Protein G is... D =1.03×10 -8 M; Protein G-M1 equilibrium dissociation constant K D The values are 1.31 × 10⁻⁶. -8 M. The affinity of Protein G-M1 for the F(ab′)2 fragment of equine serum antibody is close to that of wild-type Protein G. Therefore, it is believed that the effect of the mutation of Protein G-M1 on the affinity of the F(ab′)2 fragment is negligible, providing a theoretical basis for the subsequent purification of the F(ab′)2 fragment using Protein G-M1 affinity packing material.
[0065] Example 3
[0066] The preparation of Protein G-M1 affinity filler includes the following steps:
[0067] Protein G-M1 buffer (sodium carbonate buffer at pH 9.5) was mixed with epoxy pre-activated agarose gel (purchased from Wuhan Jingcheng, particle size 45-165 μm) and incubated at 37 °C for 8 h. This allowed the epoxy groups on the epoxy pre-activated agarose gel to form covalent bonds with the amino groups of Protein G-M1, thus achieving coupling. Then, the uncoupled activated groups were blocked with ethanolamine. The mixture was repeatedly washed with washing buffer to remove excess ligands (i.e., Protein G-M1) that had not formed covalent bonds, thus obtaining the Protein G-M1 affinity filler.
[0068] The Protein G-M1 affinity packing material was packed into the shell of the chromatography column to obtain the Protein G-M1 affinity column (abbreviated as G-M1 affinity chromatography column).
[0069] Example 4
[0070] Protein G-M1 affinity packing material specifically purifies horse serum antibody F(ab′)2 fragment.
[0071] A mixture of F(ab′)2 and Fc fragments was obtained by direct digestion of full-length IgG antibody against horse serum with pepsin;
[0072] The pH of the above mixture was adjusted to 6-7 using PBS buffer, and then loaded onto the G-M1 affinity chromatography column prepared in Example 3 using a peristaltic pump. The protein was eluted using a protein purifier, with the elution buffer for the F(ab′)2 fragment being 0.1M acetic acid aqueous solution (pH = 3.0). The results are as follows: Figure 3 As shown.
[0073] Figure 3 The images show the SDS-PAGE results of proteins purified from horse serum antibody F(ab′)2 and Fc fragments using a Protein G-M1 affinity column. In the images, A shows the purification results of the affinity column on an AKTA protein purifier, B shows the SDS-PAGE results, 1 shows the elution product, 2 shows the substance obtained from column flow-through, and 3 shows the mixture before column purification.
[0074] Depend on Figure 3 As can be seen from A, the F(ab′)2 fragment was successfully eluted in 0.1M acetic acid aqueous solution (pH=3.0);
[0075] Depend on Figure 3 B shows that lane 1 successfully obtained the purified product horse serum antibody F(ab′)2 fragment. During SDS-PAGE, the sample was not denatured, so the F(ab′)2 fragment was not broken into F(ab) and still maintained a molecular weight of 90kDa, corresponding to the results of lane 1 and lane 3.
[0076] The substance obtained in lane 2 was the Fc fragment of horse serum antibody. After being digested by pepsin, the Fc fragment was degraded into a 5-20 kDa fragment, corresponding to the results of lanes 2 and 3.
[0077] Experiments have shown that the G-M1 affinity column can specifically enrich the horse serum antibody F(ab′)2 fragment without binding to the horse serum antibody Fc fragment, thus achieving the goal of efficient and high-purity separation of the horse serum antibody F(ab′)2 and Fc fragments.
[0078] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.
Claims
1. A mutant streptococcal protein G, characterized in that, The amino acid sequence of the mutant protein G is shown below: ① TTYKLVINGKTLKGETTTKAVDAETAX1X2AFKQYANDNGVDGVWTYDDATKTFTVTE; where X1 is independently selected from one of K, R, and H; and X2 is independently selected from one of E and D. ② An amino acid sequence that has more than 80% homology with the amino acid sequence shown in ①, and still has affinity for the F(ab')2 fragment but no affinity for the Fc fragment.
2. The mutant streptococcal protein G according to claim 1, characterized in that, The mutant streptococcal protein G has an affinity for the F(ab')2 fragment but no affinity for the Fc fragment.
3. The mutant streptococcal protein G according to claim 2, characterized in that, The F(ab')2 fragment is the F(ab')2 fragment of equine serum antibody, and the Fc fragment is the Fc fragment of equine serum antibody.
4. A polynucleotide, characterized in that, The polynucleotide encodes the mutant streptococcal protein G as described in any one of claims 1-3.
5. An expression carrier, characterized in that, The expression vector contains the polynucleotide of claim 4.
6. A host cell, characterized in that, The host cell contains the expression vector of claim 5 or the host cell genome integrates the polynucleotide of claim 4; preferably, the host cell is a cell capable of expressing exogenous proteins; preferably, the host cell is a bacterium, yeast, insect cell or mammalian cell.
7. A fusion protein, characterized in that, The fusion protein comprises: the mutant streptococcal protein G as described in any one of claims 1-3, and has a 6×His tag attached to the C-terminus of the amino acid sequence of the mutant streptococcal protein G.
8. An adsorbent material, characterized in that, The mutant streptococcal protein G of any one of claims 1-3 or the fusion protein of claim 7 is immobilized on a carrier to obtain an adsorbent material; preferably, the carrier is agarose; preferably, the agarose is pre-activated with epoxy.
9. The use of the mutant streptococcal protein G as described in any one of claims 1-3, the fusion protein as described in claim 7, and the adsorption material as described in claim 8 in the separation and purification of F(ab')2 fragments and their derivatives, and as packing material for chromatography columns; preferably, the F(ab')2 fragment is the F(ab')2 fragment of horse serum antibody.
10. A method for separating and purifying F(ab')2 fragments, characterized in that, The method includes the following steps: using the adsorbent material described in claim 8 as the column packing material, performing column chromatography on a sample containing the F(ab')2 fragment to obtain the F(ab')2 fragment; preferably, the F(ab')2 fragment is the F(ab')2 fragment of horse serum antibody; preferably, the eluent for column chromatography is an aqueous acetic acid solution; preferably, the pH of the aqueous acetic acid solution is 2.8-3.2.