A polypeptide, multimer and use thereof in immunoglobulin affinity ligands

Abstract

The present application relates to the technical field of biological medicine, and proposes a kind of polypeptide, multimer and its application in immunoglobulin affinity ligand, the polypeptide includes the 49-58 amino acids of the B domain of protein A sequence, obtain the truncated body sequence shown in SEQ ID NO:2, while replacing all lysine of the truncated body to generate a series of amino acid sequences.The multimer connected by the polypeptide of the present application can be used as immunoglobulin affinity ligand, which can bind more immunoglobulin per unit weight or unit volume of carrier, and improve the binding capacity of immunoglobulin.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a polypeptide, a polymer, and its application in immunoglobulin affinity ligands. Background Technology

[0002] Natural proteins consist of 20 amino acid residues, the properties of which are influenced by the properties of their side chain functional groups. Typically, proteins are chemically bonded using reactions of their side chains on insoluble carriers or similar substances, attempting to immobilize them. However, many naturally derived proteins have far more than one hundred amino acid residues; if we focus on a specific amino acid residue, multiple amino acid residues exist within each protein molecule. This is one of the main reasons why it is difficult to control the reaction when using the functional groups of specific amino acids for immobilization and chemical modification.

[0003] Protein A, derived from Staphylococcus aureus, possesses five immunoglobulin-binding domains (E, D, A, B, and C). Each of these domains can bind immunoglobulins independently and contains lysine (Lys, K) but not cysteine ​​(Cys). Domain B is widely used due to its high specificity in binding to immunoglobulins. Mutating the alanine at position 1 and the glycine at position 29 of domain B to valine and alanine, respectively, yields the derived Z domain, which exhibits superior chemical stability. Currently, protein A variants generated through recombinant protein engineering and native protein A are widely used as ligands immobilized on water-insoluble carriers.

[0004] Immobilizing ligands such as protein A onto a carrier generates a medium capable of binding immunoglobulins. A high immunoglobulin binding capacity per unit weight or volume of this medium is crucial for reducing the cost of immunoglobulin purification. Improving the performance of immunoglobulin-binding proteins is an important aspect of addressing this issue. When immobilizing protein ligands, including protein A, onto a carrier, single-site immobilization is advantageous in terms of allowing for directional control and efficient utilization of the carrier surface area. Most existing techniques involve adding Cys sites to the protein ends, but proteins with Cys sites at the ends tend to form dimers. Given this problem, one problem this invention aims to solve is to develop a protein A ligand for terminal Lys site-specific immobilization techniques, providing an affinity separation medium with high immunoglobulin binding capacity. Summary of the Invention

[0005] In view of this, the present invention proposes a polypeptide, a polymer and its application in immunoglobulin affinity ligands, wherein the polymer can be used as an affinity chromatography medium and has a high binding capacity for immunoglobulins.

[0006] The technical solution of the present invention is implemented as follows: In a first aspect, the present invention provides a polypeptide, the polypeptide comprising the amino acid sequence shown in SEQ ID NO:2 after truncation of amino acid sequences at positions 49-58 of the B domain of protein A shown in SEQ ID NO:1.

[0007] Based on the above technical solutions, preferably, the amino acid sequence shown in SEQ ID NO:2 has substitution mutations at the Lys residues at positions 4, 7 and 35.

[0008] Based on the above technical solution, preferably, the Lys residues at positions 4, 7 and 35 are replaced with one or more of glycine, phenylalanine, asparagine, valine, histidine, alanine, aspartic acid, leucine, threonine, tyrosine, serine and arginine.

[0009] Specifically, the three Lys residues at positions 4, 7, and 35 are either replaced by different amino acids or by the same amino acid.

[0010] Based on the above technical solution, preferably, the three Lys residues at positions 4, 7 and 35 are all replaced by glycine, and the amino acid sequence is shown in SEQ ID NO:3.

[0011] Based on the above technical solution, preferably, the three Lys residues at positions 4, 7 and 35 are all replaced by arginine, and the amino acid sequence is shown in SEQ ID NO:4.

[0012] In a second aspect, the present invention also provides a polymer comprising 2-6 linked polypeptides, wherein the polypeptides are any one of the polypeptides shown in SEQ ID NO:2-4.

[0013] Based on the above technical solutions, preferably, peptides are directly linked by C-terminal and N-terminal peptide bonds or linked by a linker.

[0014] The linker is a conventional linker used in the art, the presence of which does not affect the binding activity of the peptide. Exemplary linkers are linker sequences containing G and S or composed of G and S, typically 2-20 amino acid residues in length. Exemplary linker sequences include (G)n, (GS)n, (GSS)n, (GSSS)n, (GSSSS)n, and (GGGGS)n, where n can be an integer from 2 to 10, but the total length of the linker typically does not exceed 20 amino acid residues.

[0015] Based on the above technical solutions, preferably, the amino acid sequence of the polymer is modified by adding 1-10 Lys residues to the end.

[0016] Based on the above technical solutions, preferably, the amino acid sequence of the polymer may or may not contain a tag.

[0017] The tags are those commonly used in the art, and their presence does not affect the binding activity of the multimer. Exemplary tags include His-tag (histidine tag), GST-tag (glutathione thiotransferase tag), MBP-tag (maltose-binding protein tag), SUMO-tag (small molecule ubiquitin-like modified protein tag), etc.

[0018] Based on the above technical solutions, preferably, the amino acid sequences of the polymer are shown in SEQ ID NO:5~7, SEQ ID NO:15~17 and SEQ ID NO:25~27.

[0019] Thirdly, the present invention also provides the application of a multimer in immunoglobulin affinity ligands.

[0020] The polypeptide, polymer, and their application in immunoglobulin affinity ligands of the present invention have the following advantages over the prior art:

[0021] This invention provides an immunoglobulin affinity ligand with high immunoglobulin binding capacity. By modifying the B domain of protein A shown in SEQ ID NO:1, firstly, amino acids at positions 49-58 are truncated to generate a truncated variant. Then, all Lys residues in the truncated variant are mutated by substitution. Finally, the lysine mutant of the truncated variant is repeatedly tandemly repeated to construct a multimer, with 1-10 Lys residues added to the end of the multimer as coupling elements. The modified ligand can be immobilized on an insoluble carrier via the amino groups of the coupling elements. Compared to the truncated variant, the orientation of the lysine mutant on the carrier is improved, allowing more immunoglobulins to bind per unit weight or volume of carrier, thereby enhancing the immunoglobulin binding capacity. The advantage of this invention is that it improves ligand orientation and enhances the immunoglobulin binding capacity of the ligand. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1The image shows the amino acid alignment of the five homologous domains (E, D, A, B, and C) of protein A. The positions of helix 1, helix 2, and helix 3 are 7-18, 25-36, and 41-54, respectively. The lysine residues in the five domains are highlighted in yellow in the attached image.

[0024] Figure 2 To demonstrate the relationship between the amount of ligand immobilization and the antibody binding ability of the affinity separation medium, I represents proteins of prototypes 1-3, II represents proteins of prototypes 4-6, and III represents proteins of prototypes 7-9. Detailed Implementation

[0025] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0026] Protein A is a protein derived from the cell wall of Staphylococcus aureus. It consists of five immunoglobulin-binding domains, each of which is composed of three α-helical structures and two loop structures that connect the three α-helical structures to each other.

[0027] The main active groups in proteins that couple with solid carriers include the thiol group of cysteine, the amino group of N-terminal amino acids and Lys, the carboxyl group of C-terminal amino acids and glutamic acid, and the carboxyl group of aspartic acid. The inventors of this invention focused on the Lys residue, as Lys residues at different positions in the protein can bind to the carrier, and Lys residues are suitable for controlling the coupling direction.

[0028] This invention investigates amino acid truncation and mutation. First, the Lys residue and the amino acid sequence following it in the B domain helix 3 are truncated, and then all Lys residues in the remaining sequence are replaced by mutation.

[0029] In a first aspect, the present invention discloses a polypeptide that, compared with the native B domain shown in SEQ ID NO:1, has its amino acid sequence truncated from positions 49 to 58 to obtain the truncated sequence shown in SEQ ID NO:2; and then has Lys substitution mutations at all three positions at positions 4, 7, and 35. The underlined regions indicate the truncated areas.

[0030]

[0031] In "amino acid substitution of Lys residues," the types of amino acids introduced through substitution include, but are not limited to, natural amino acids, non-protein amino acids, and non-natural amino acids. From the perspective of production through genetic engineering, natural amino acids can be appropriately used.

[0032] The three Lys residues at positions 4, 7, and 35 are simultaneously substituted with one or more of the following: glycine, phenylalanine, asparagine, valine, histidine, alanine, aspartic acid, leucine, threonine, tyrosine, serine, and arginine.

[0033] In this invention, the three Lys residues at positions 4, 7, and 35 were all replaced with glycine (Gly, G), resulting in the amino acid sequence shown in SEQ ID NO:3. The bold underlined amino acids are mutant amino acids.

[0034] In another embodiment, the present invention selects to replace the three Lys residues at positions 4, 7 and 35 with arginine (Arg, R) to obtain the amino acid sequence shown in SEQ ID NO:4.

[0035] amino acid sequence SEQID ADNGFNGEQQNAFYEILHLPNLNEEQRNGFIQSLGDDPSQSANLLAEA NO3 ADNRFNREQQNAFYEILHLPNLNEEQRNGFIQSLRDDPSQSANLLAEA NO4

[0036] The following description is provided to illustrate the invention in more detail based on examples, but the scope of the invention is not limited to these examples. The proteins obtained in the examples are each represented by "letter representing the domain - introduced mutation (wild represents wild type)". For example, the wild-type B domain of protein A is represented by "B-wild", the B domain retaining 48 amino acid residues is represented by "B48", and the B domain variant containing the K04R mutation while retaining 48 amino acid residues is represented by "B48-K04R". A B48 domain variant containing two mutations is represented by slashes indicating the two mutations. For example, a B domain variant containing both K04R and K07R mutations is represented by "B48-K04R / K07R". For proteins containing multiple linked monomeric domains, the number of connected domains is further indicated by periods (.) and "d". For example, a protein consisting of three linked B48 domain variants containing K04R, K07R, and K35R mutations is represented as “B48-K04R / K07R / K35R.3d”.

[0037] Example 1: Preparation of ligand expression plasmid

[0038] I. Design of affinity ligands based on the truncated B domain (B48.1d, SEQ ID NO 2).

[0039] The following affinity ligands were used in this embodiment:

[0040] (1)B48.2d (SEQ ID NO 5), which connects two such structural domains;

[0041] (2)B48.3d (SEQ ID NO 6), which connects three such structural domains;

[0042] (3)B48.4d(SEQ ID NO 7), which connects 4 such structural domains.

[0043]

[0044] Taking B48.2d (SEQ ID NO 5) as an example, B48.2d (SEQ ID NO 5) is a truncated form of 48 amino acids with two B domains. The C-terminus contains three Lys as coupling elements, and the N-terminus contains hexahistine as a purification tag. It is inserted into the expression vector pET-28a through the 5' NcoⅠ and 3' XhoⅠ restriction sites. The complete DNA sequence (SEQ ID NO 8) was synthesized by a third-party gene company.

[0045] Using the pET-28a vector containing the sequence of SEQ ID NO 8 as a template, Overlap PCR was performed with primers SEQ ID NO 9 / SEQ ID NO 10 and SEQ ID NO 11 / SEQ ID NO 12 to amplify the DNA sequences encoding B48.3d (SEQ ID NO 13, containing NcoⅠ / XhoⅠ restriction sites) and B48.4d (SEQ ID NO 14, containing NcoⅠ / XhoⅠ restriction sites). The target fragments were then ligated to the pET-28a double-digested vector using homologous recombinase. The DNA sequences of B48.3d (SEQ ID NO 13) and B48.4d (SEQ ID NO 14) were sequenced and verified by a third-party gene company.

[0046]

[0047] II. Affinity ligands were designed based on the mutant of the B-domain truncated form (B48-K04G / K07G / K35G.1d, SEQ ID NO 3), in which all Lys residues were replaced by Gly residues.

[0048] The following affinity ligands were used in this embodiment:

[0049] (1)B48-K04G / K07G / K35G.2d(SEQ ID NO 15), which connects two such structural domains;

[0050] (2)B48-K04G / K07G / K35G.3d(SEQ ID NO 16), which connects 3 such structural domains;

[0051] (3)B48-K04G / K07G / K35G.4d(SEQ ID NO 17), which connects 4 such structural domains.

[0052]

[0053]

[0054] Taking B48-K04G / K07G / K35G.2d (SEQ ID NO 15) as an example, B48-K04G / K07G / K35G.2d (SEQ ID NO 15) is a mutant with two truncated B domains. The C-terminus contains three Lys as coupling elements, and the N-terminus contains hexahistine as a purification tag. It is inserted into the expression vector pET-28a through the 5' NcoⅠ and 3' XhoⅠ restriction sites. The complete DNA sequence (SEQ ID NO 18) was synthesized by a third-party gene company.

[0055] Using the pET-28a vector containing the sequence of SEQ ID NO 18 as a template, Overlap PCR was performed with primers SEQ ID NO 19 / SEQ ID NO 20 and SEQ ID NO 21 / SEQ ID NO 22 to amplify the DNA sequences encoding B48-K04G / K07G / K35G.3d (SEQ ID NO 23, containing NcoⅠ / XhoⅠ restriction sites) and B48-K04G / K07G / K35G.4d (SEQ ID NO 24, containing NcoⅠ / XhoⅠ restriction sites). The target fragments were then ligated to the pET-28a double-digested vector using homologous recombinase. The DNA sequences of B48-K04G / K07G / K35G.3d (SEQ ID NO 23) and B48-K04G / K07G / K35G.4d (SEQ ID NO 24) were sequenced and verified by a third-party gene company.

[0056]

[0057]

[0058] III. Affinity ligands were designed based on the mutant of the B domain truncated form (B48-K04R / K07R / K35R.1d, SEQ ID NO 4), in which all Lys residues were replaced by Arg residues.

[0059] The following affinity ligands were used in this embodiment:

[0060] (1)B48-K04R / K07R / K35R.2d(SEQ ID NO 25), which connects two such structural domains;

[0061] (2)B48-K04R / K07R / K35R.3d(SEQ ID NO 26), which connects three such structural domains;

[0062] (3)B48-K04R / K07R / K35R.4d(SEQ ID NO 27), which connects four such structural domains.

[0063]

[0064]

[0065] Taking B48-K04G / K07G / K35G.2d (SEQ ID NO 25) as an example, B48-K04G / K07G / K35G.2d (SEQ ID NO 25) is a mutant with two truncated B domains. The C-terminus contains three Lys as coupling elements, and the N-terminus contains hexahistine as a purification tag. It is inserted into the expression vector pET-28a through the 5' NcoⅠ and 3' XhoⅠ restriction sites. The complete DNA sequence (SEQ ID NO 28) was synthesized by a third-party gene company.

[0066] Using the pET-28a vector containing the sequence of SEQ ID NO 28 as a template, Overlap PCR was performed with primers SEQ ID NO 19 / SEQ ID NO 29 and SEQ ID NO 30 / SEQ ID NO 22 to amplify the DNA sequences encoding B48-K04R / K07R / K35R.3d (SEQ ID NO 31, containing NcoⅠ / XhoⅠ restriction sites) and B48-K04R / K07R / K35R.4d (SEQ ID NO 32, containing NcoⅠ / XhoⅠ restriction sites). The target fragments were then ligated to the pET-28a double-digested vector using homologous recombinase.

[0067] The DNA sequences of B48-K04R / K07R / K35R.3d (SEQ ID NO 31) and B48-K04R / K07R / K35R.4d (SEQ ID NO 32) were sequenced and verified by a third-party gene company.

[0068]

[0069]

[0070] Table 1B shows the polymers of domain mutants.

[0071] Target protein number Target protein plasmid source prototype1 B48.2d Genetic synthesis commissioned to a gene company prototype2 B48-K04G / K07G / K35G.2d Genetic synthesis commissioned to a gene company prototype3 B48-K04R / K07R / K35R.2d Genetic synthesis commissioned to a gene company prototype4 B48.3d Constructed from prototype1 plasmid prototype5 B48-K04G / K07G / K35G.3d Constructed from prototype2 plasmid prototype6 B48-K04R / K07R / K35R.3d Constructed from prototype3 plasmid prototype7 B48.4d Constructed from prototype1 plasmid prototype8 B48-K04G / K07G / K35G.4d Constructed from prototype2 plasmid prototype9 B48-K04R / K07R / K35R.4d Constructed from prototype3 plasmid

[0072] Example 2: Expression and purification of ligands

[0073] The recombinant plasmid from Example 1 was transformed into BL21(DE3) Escherichia coli competent cells via heat shock, and cultured in LB medium for IPTG-induced expression. After induction, the cells were collected, and the cell walls were disrupted by sonication to release the expression product. The supernatant was collected by centrifugation. The supernatant was purified using Ni column affinity medium, and impurities were washed away with low-concentration imidazole, followed by elution of the target protein with high-concentration imidazole. The target protein eluent was dialyzed against 0.01M PBS, and then the target protein was concentrated using ultrafiltration and stored for later use.

[0074] Example 3 Evaluation of IgG binding capacity of ligand affinity chromatography medium

[0075] Following conventional methods, each immunoglobulin-binding protein purified in Example 2 was immobilized on an NHS-activated agarose gel support at different doses.

[0076] The performance of affinity chromatography media for B-domain mutants was tested using commercially available human immunoglobulin as the adsorbent. Human immunoglobulin was diluted to a specific concentration with 0.01 mol / L PBS. The adsorbent was weighed into a 10 mL centrifuge tube, and the human immunoglobulin diluent was added. The tube was then placed on a shaker at 37°C and 120 rpm for 2 h. After the adsorbent settled naturally for 30 min, the supernatant was collected for analysis. The human immunoglobulin content in the samples before and after adsorption was measured to determine the adsorption efficiency. The results are shown below. Figure 2 As shown.

[0077] Figure 2 The results show the evaluation of the immunoglobulin binding activity of the immunoglobulin-binding protein of the present invention used as an affinity ligand, which is based on the value obtained by dividing the amount of human immunoglobulin (mg) adsorbed on the chromatography medium by the amount of immobilized ligand (mg) (IgG / ligand ratio).

[0078] For dimeric ligands, Prototype 2 (a dimeric protein with a glycine mutant of the B-domain truncated form) and Prototype 3 (a dimeric protein with an arginine mutant of the B-domain truncated form) adsorbed more immunoglobulins than Prototype 1 (a dimeric protein with the B-domain truncated form) with the same fixed amount of ligand. Similarly, for trimeric ligands, Prototype 5 (a trimeric protein with a glycine mutant of the B-domain truncated form) and Prototype 6 (a trimeric protein with an arginine mutant of the B-domain truncated form) adsorbed more immunoglobulins than Prototype 4 (a trimeric protein with the B-domain truncated form) with the same fixed amount of ligand. Similarly, for tetrameric ligands, Prototype 8 (a tetrameric protein with a glycine mutant and a truncated B domain) and Prototype 9 (a tetrameric protein with an arginine mutant and a truncated B domain) adsorbed more immunoglobulins than Prototype 7 (a tetrameric protein with a truncated B domain) under the same fixed amount of ligand. These results indicate that:

[0079] I. The truncated form of SEQ ID NO 2 obtained by truncating amino acids at positions 49-58 of the B domain of protein A has immunoglobulin binding activity.

[0080] II. The three Lys in the truncated B domain sequence were mutated by replacing them with glycine. Three Lys were added as coupling elements to the ends of dimers, trimers and tetramers that did not contain Lys. Compared with the truncated version, the amount of immunoglobulin adsorbed by the glycine-substituted ligand was increased in affinity media with different ligand coupling amounts.

[0081] 3. Mutation by replacing three Lys with arginine in the truncated B domain sequence, adding three Lys as coupling elements to the ends of dimers, trimers, and tetramers that do not contain Lys, resulted in increased adsorption of immunoglobulins by the arginine-substituted ligands in affinity media with varying ligand coupling amounts compared to the truncated version.

[0082] IV. When the ligand coupling amount is less than 15 mg / g, the ligand with arginine substitution mutation has better performance than the glycine substitution mutation group and the truncated group. The affinity medium per unit weight can bind a larger amount of immunoglobulin, thus improving the immunoglobulin binding capacity.

[0083] Currently, the optimal embodiment of the immunoglobulin-binding activity of this invention involves a modified protein. First, the amino acid sequence at positions 49-58 of the B domain of protein A is truncated to obtain a truncated form. Then, the three Lys residues at positions 4, 7, and 35 of the truncated form are mutated by substitution mutations of other amino acids. Multiple tandemly expressed truncated polypeptides are then expressed, and lysine residues are added to the terminal of the multimeric protein as coupling sites. As an affinity mediator, the modified protein described in this patent exhibits immunoglobulin-binding activity, and it has been found that its immunoglobulin-binding activity is significantly enhanced compared to the truncated protein without the Lys mutations at positions 4, 7, and 35. Furthermore, the modified protein A maintains good orientation when immobilized on a solid support via Lys residues. In other words, the modified protein of this invention possesses structural and functional properties highly useful as an affinity ligand for immunoglobulins.

[0084] It should be noted that simultaneously introducing the above changes into the Z-domain is also within the scope of protection of this patent.

[0085] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A polypeptide, characterized in that: The amino acid sequence of the polypeptide is shown as SEQ ID NO:

2.

2. A polypeptide, characterized in that: The amino acid sequence of the polypeptide is shown as SEQ ID NO:

3.

3. A polypeptide, characterized in that: The amino acid sequence of the polypeptide is shown as SEQ ID NO:

4.

4. A multimer, characterized by: The multimer comprises 2-6 linked polypeptides, the polypeptides being the polypeptide of any one of claims 1-3.

5. A multimer according to claim 4, wherein: - the first and second polypeptides are identical; and - the third and fourth polypeptides are identical. The polypeptides are connected by peptide bonds at the C- and N-termini, respectively, directly or via a linker.

6. A multimer according to claim 4, wherein: The modification of adding 1-10 lysine residues at the end of the amino acid sequence of the multimer.

7. A multimer according to claim 4, wherein: The amino acid sequence of the multimer is shown as SEQ ID NO: 5-7, SEQ ID NO: 15-17 and SEQ ID NO: 25-27.

8. Use of a multimer according to any one of claims 4-7 for the manufacture of an immunoglobulin affinity ligand.

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