Polypeptide, polymer and application thereof in immunoglobulin affinity ligand

Abstract

The invention relates to the technical field of biological medicine, and provides a polypeptide, a polymer and application of the polypeptide in an immunoglobulin affinity ligand.The polypeptide comprises 49-58 amino acid of a B structural domain of a protein A sequence, a truncated body sequence shown in SEQ ID NO: 2 is obtained, and meanwhile a series of amino acid sequences generated by all lysine of the truncated body are replaced. A polymer formed by polypeptide connection can be used as an immunoglobulin affinity ligand, so that a carrier in unit weight or unit volume can be combined with more immunoglobulins, and the binding capacity to the immunoglobulins is improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a polypeptide, a polymer and the application thereof in immunoglobulin affinity ligands. Background Art

[0002] Natural proteins are composed of 20 kinds of amino acid residues, and the properties of each amino acid residue are affected by the properties of its side chain functional groups. Usually, proteins are chemically bonded using the reaction of side chains on insoluble carriers or analogs and are attempted to be immobilized. However, the number of amino acid residues in many natural proteins is far more than 100, and if we focus on a specific amino acid residue, there are multiple amino acid residues in 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 is derived from Staphylococcus aureus and has five immunoglobulin binding domains (E, D, A, B, C). These domains can bind to immunoglobulins individually and contain lysine (Lys, K) but no cysteine ​​(Cys). The B domain is widely used because it has good binding specificity with immunoglobulins. The Z domain is derived by mutating the alanine at position 1 and the glycine at position 29 in the B domain to valine and alanine, respectively. The Z domain has more outstanding chemical stability. At present, protein A variants and natural protein A produced by recombinant protein engineering technology are widely used as ligands to be fixed on water-insoluble carriers.

[0004] Fixing ligands such as protein A on a carrier generates a medium that can bind to immunoglobulins. The medium per unit weight or unit volume should have a large immunoglobulin binding capacity. This property is crucial to reducing the cost of immunoglobulin purification. In order to solve this problem, improving the performance of immunoglobulin binding proteins is an important part. When protein ligands including protein A are fixed to a carrier, single-site fixation is advantageous in allowing control of direction and effective use of the carrier surface area. Most of the existing technologies currently add Cys sites to the ends of proteins, and proteins containing Cys sites at the ends are prone to form dimers. In view of this problem, one problem to be solved by the present invention is to develop a protein A ligand for terminal Lys site-specific fixation technology to provide an affinity separation medium with high binding ability to immunoglobulins. Summary of the invention

[0005] In view of this, the present invention proposes a polypeptide, a polymer and the use of the same in an immunoglobulin affinity ligand. 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 the first aspect, the present invention provides a polypeptide, which includes the amino acid sequence shown in SEQ ID NO:2 after the amino acid sequence at positions 49-58 in the B domain of protein A shown in SEQ ID NO:1 is truncated.

[0007] On the basis of the above technical solution, preferably, the amino acid sequence shown in SEQ ID NO: 2 undergoes substitution mutations at the Lys residues at positions 4, 7 and 35.

[0008] On the basis of the above technical scheme, preferably, the Lys residues at positions 4, 7 and 35 are replaced by 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 substituted by different amino acids or by the same amino acid.

[0010] On the basis of 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] On the basis of 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 further provides a multimer, wherein the multimer comprises 2-6 linked polypeptides, wherein the polypeptide is any one of the polypeptides shown in SEQ ID NOs: 2-4.

[0013] On the basis of the above technical solution, preferably, the polypeptides are directly connected via peptide bonds at the C-terminus and the N-terminus or connected via a linker.

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

[0015] On the basis of the above technical solution, preferably, the terminal of the amino acid sequence of the polymer is modified by adding 1-10 Lys residues.

[0016] On the basis of the above technical solution, preferably, the amino acid sequence of the polymer contains or does not contain a tag.

[0017] The tag is a tag commonly used in the art, and its presence does not affect the binding activity of the polymer. Exemplary tags include His-tag (histidine tag), GST-tag (glutathione sulfhydryl transferase tag), MBP-tag (maltose binding protein tag), SUMO-tag (small molecule ubiquitin-like modified protein tag), etc.

[0018] On the basis of the above technical solution, preferably, the amino acid sequence of the polymer is as shown in SEQ ID NOs: 5 to 7, SEQ ID NOs: 15 to 17 and SEQ ID NOs: 25 to 27.

[0019] In a third aspect, the present invention also provides a use of a multimer in an immunoglobulin affinity ligand.

[0020] The polypeptide, polymer and application thereof in immunoglobulin affinity ligand of the present invention have the following beneficial effects compared with the prior art:

[0021] The present invention provides an immunoglobulin affinity ligand having a high immunoglobulin binding ability. By modifying the B domain of protein A shown in SEQ ID NO: 1, first, the amino acids at positions 49-58 are truncated to generate a truncate, then all Lys in the truncate are mutated by substitution, and finally the lysine mutants of the truncate are repeated multiple times in tandem to construct a multimer and 1-10 Lys are added to the end of the multimer as a coupling element. The modified ligand can be fixed on an insoluble carrier through the amino group of the coupling element. Compared with the truncate, the orientation of the lysine mutant on the carrier is improved, and the carrier per unit weight or unit volume can bind more immunoglobulins, thereby improving the binding ability with the immunoglobulin. The advantages of the present invention are that the orientation of the ligand can be improved and the immunoglobulin binding ability of the ligand can be improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1The figure is an amino acid alignment diagram 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 marked in yellow in the figure.

[0024] Figure 2 To show the relationship between the amount of ligand fixed and the antibody binding capacity of the affinity separation medium, Ⅰ is prototype 1 to 3 target proteins, Ⅱ is prototype 4 to 6 target proteins, and Ⅲ is prototype 7 to 9 target proteins. DETAILED DESCRIPTION

[0025] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] The protein A molecule is a protein derived from the cell wall of Staphylococcus aureus, and includes five immunoglobulin binding domains, each of which is composed of three α-helical structures and two loop structures connecting the three α-helical structures to each other.

[0027] The main active groups in proteins that couple with solid supports include the sulfhydryl group of cysteine, the amino group of the N-terminal amino acid and Lys, the carboxyl group of the C-terminal amino acid and glutamic acid, aspartic acid, etc. The inventors of the present invention focus on Lys residues, as Lys residues at different positions in proteins can bind to supports, and Lys residues are a type of residue suitable for controlling the direction of coupling.

[0028] The present invention studies amino acid truncation and mutation, firstly truncating the Lys residue in helix 3 of the B domain and the amino acid sequence thereafter, and then replacing all Lys residues in the remaining sequence with mutations.

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

[0030]

[0031] In "amino acid substitution of Lys residues", the types of amino acids introduced by substitution include, but are not limited to, natural amino acids, non-protein amino acids, and non-natural amino acids. From the viewpoint of production by 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 glycine, phenylalanine, asparagine, valine, histidine, alanine, aspartic acid, leucine, threonine, tyrosine, serine and arginine.

[0033] The present invention selects to replace the three Lys residues at positions 4, 7 and 35 with glycine (Gly, G) to obtain the amino acid sequence shown in SEQ ID NO: 3. The bold underlined amino acids are mutated 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 SEQ ID ADNGFNGEQQNAFYEILHLPNLNEEQRNGFIQSLGDDPSQSANLLAEA NO3 ADNRFNREQQNAFYEILHLPNLNEEQRNGFIQSLRDDPSQSANLLAEA NO4

[0036] The following description is provided to illustrate the present invention in more detail based on examples, but the scope of the present invention is not limited to these examples. The proteins obtained in the embodiments are each represented by "letters representing domains-introduced mutations (wild represents wild type)". For example, the wild-type B domain of protein A is represented by "B-wild", the B domain with 48 amino acid residues is represented by "B48", and the B domain variant with 48 amino acid residues and K04R mutation is represented by "B48-K04R". The B48 domain variant containing two mutations at the same time is represented by two mutations represented by slashes. For example, the B domain variant containing K04R and K07R mutations is represented by "B48-K04R / K07R". For proteins containing multiple monomer domains connected together, the number of connected domains with a period (.) and "d" is further represented. For example, a protein consisting of three linked B48 domain variants comprising K04R, K07R, and K35R mutations is represented by "B48-K04R / K07R / K35R.3d."

[0037] Example 1 Preparation of ligand expression plasmid

[0038] 1. Design of affinity ligand based on B domain truncation (B48.1d, SEQ ID NO 2).

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

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

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

[0042] (3) B48.4d (SEQ ID NO 7), in which four such domains are linked.

[0043]

[0044] Taking B48.2d (SEQ ID NO 5) as an example, B48.2d (SEQ ID NO 5) connected two 48-amino acid truncations of the B domain, contained three Lys at the C-terminus as coupling elements, and contained hexahistidine as a purification tag at the N-terminus. It was inserted into the expression vector pET-28a through the 5'-end NcoⅠ and 3'-end XhoⅠ restriction sites, and the complete DNA sequence (SEQ ID NO 8) was commissioned to be synthesized by a third-party gene company.

[0045] Using the pET-28a vector containing SEQ ID NO 8 as a template, SEQ ID NO 9 / SEQ ID NO 10 and SEQ ID NO 11 / SEQ ID NO 12 primers were used for Over-lap PCR to amplify the DNA sequence encoding B48.3d (SEQ ID NO 13, containing NcoⅠ / XhoⅠ restriction sites) and B48.4d (SEQ ID NO 14, containing NcoⅠ / XhoⅠ restriction sites), and the target fragment and the pET-28a double restriction vector were connected with homologous recombinase. The DNA sequence of B48.3d (SEQ ID NO 13) and the DNA sequence of B48.4d (SEQ ID NO 14) were entrusted to a third-party gene company for sequencing verification.

[0046]

[0047] Second, an affinity ligand was designed based on a mutant of the B domain truncation (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 example:

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

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

[0051] (3) B48-K04G / K07G / K35G.4d (SEQ ID NO 17), in which four such domains are linked.

[0052]

[0053]

[0054] Taking B48-K04G / K07G / K35G.2d (SEQ ID NO 15) as an example, B48-K04G / K07G / K35G.2d (SEQID NO 15) is connected with two mutants of B domain truncations, contains 3 Lys at the C-terminus as coupling elements, and contains hexahistidine as a purification tag at the N-terminus. It is inserted into the expression vector pET-28a through the 5'-end NcoⅠ and 3'-end XhoⅠ restriction sites. The complete DNA sequence (SEQ ID NO 18) was commissioned to be synthesized by a third-party gene company.

[0055] Using the pET-28a vector containing SEQ ID NO 18 as a template, SEQ ID NO 19 / SEQ ID NO 20 and SEQ ID NO 21 / SEQ ID NO 22 primers were used for Over-lap PCR to amplify the DNA sequence encoding B48-K04G / K07G / K35G.3d (SEQ ID NO 23, containing NcoⅠ / XhoⅠ restriction sites) and the DNA sequence of B48-K04G / K07G / K35G.4d (SEQ ID NO 24, containing NcoⅠ / XhoⅠ restriction sites), and the target fragment and the pET-28a double restriction vector were connected with homologous recombinase. The DNA sequence of B48-K04G / K07G / K35G.3d (SEQ ID NO 23) and the DNA sequence of B48-K04G / K07G / K35G.4d (SEQ ID NO 24) were entrusted to a third-party gene company for sequencing verification.

[0056]

[0057]

[0058] 3. Affinity ligands were designed based on a mutant of the B domain truncation (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 example:

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

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

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

[0063]

[0064]

[0065] Taking B48-K04G / K07G / K35G.2d (SEQ ID NO 25) as an example, B48-K04G / K07G / K35G.2d (SEQID NO 25) is connected with two mutants of B domain truncations, contains 3 Lys at the C-terminus as coupling elements, and contains hexahistidine as a purification tag at the N-terminus. It is inserted into the expression vector pET-28a through the 5'-end NcoⅠ and 3'-end XhoⅠ restriction sites. The complete DNA sequence (SEQ ID NO 28) was commissioned to be synthesized by a third-party gene company.

[0066] Using the pET-28a vector containing SEQ ID NO 28 as a template, Over-lap PCR was performed with primers SEQ ID NO 19 / SEQ ID NO29 and SEQ ID NO 30 / SEQ ID NO 22 to amplify the DNA sequence encoding B48-K04R / K07R / K35R.3d (SEQ ID NO 31, containing NcoⅠ / XhoⅠ restriction site) and the DNA sequence of B48-K04R / K07R / K35R.4d (SEQ ID NO 32, containing NcoⅠ / XhoⅠ restriction site), and the target fragment and the pET-28a double restriction vector were ligated with homologous recombinase.

[0067] The DNA sequence of B48-K04R / K07R / K35R.3d (SEQ ID NO 31) and the DNA sequence of B48-K04R / K07R / K35R.4d (SEQ ID NO 32) were commissioned to a third-party genetic company for sequencing verification.

[0068]

[0069]

[0070] Table 1 Multimers of B domain mutants

[0071] Target protein number Target protein Plasmid source prototype1 B48.2d Commissioned gene company to synthesize prototype2 B48-K04G / K07G / K35G.2d Commissioned gene company to synthesize prototype3 B48-K04R / K07R / K35R.2d Commissioned gene company to synthesize 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 ligand

[0073] The recombinant plasmid in Example 1 was transformed into BL21 (DE3) Escherichia coli competent cells by heat shock method, and cultured by LB medium and induced by IPTG. After induced expression, the bacteria were collected and the cell wall was destroyed by ultrasonic lysis to release the expression product and the supernatant was collected by centrifugation. The supernatant was purified by Ni column affinity medium, and the impurities were washed with low concentration of imidazole, and the target protein was eluted with high concentration of imidazole. The target protein eluate was dialyzed with 0.01M PBS, and then the target protein was concentrated by ultrafiltration tube and stored for use.

[0074] Example 3 Evaluation of IgG Binding Capacity of Ligand Affinity Chromatography Media

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

[0076] Commercial human immunoglobulin was used as the adsorption object to detect the performance of the B domain mutant affinity chromatography medium. Human immunoglobulin was diluted to a certain concentration with 0.01 mol / L PBS, the adsorbent was weighed in a 10 mL centrifuge tube, and the human immunoglobulin diluent was added. The tube was placed on a constant temperature shaker at 37 ° C and shaken at 120 r / min for 2 hours. After the adsorbent was naturally settled for 30 minutes, the supernatant was aspirated for testing. The human immunoglobulin content in the sample before and after adsorption was determined to determine the adsorption efficiency. The results are as follows: Figure 2 shown.

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

[0078] For dimeric ligands, Prototype 2 (dimeric protein of glycine mutant of B domain truncation) and Prototype 3 (dimeric protein of arginine mutant of B domain truncation) have greater amounts of immunoglobulin adsorbed than Prototype 1 (dimeric protein of B domain truncation) at the same amount of ligand immobilization. Similarly, for trimeric ligands, Prototype 5 (trimeric protein of glycine mutant of B domain truncation) and Prototype 6 (trimeric protein of arginine mutant of B domain truncation) have greater amounts of immunoglobulin adsorbed than Prototype 4 (trimeric protein of B domain truncation) at the same amount of ligand immobilization. Similarly, for tetrameric ligands, Prototype 8 (tetrameric protein of glycine mutant of B domain truncation) and Prototype 9 (tetrameric protein of arginine mutant of B domain truncation) both adsorbed more immunoglobulin than Prototype 7 (tetrameric protein of B domain truncation) at the same ligand immobilization amount. These results indicate that:

[0079] 1. The truncation of amino acids 49-58 of the B domain of protein A to obtain SEQ ID NO 2 has immunoglobulin binding activity.

[0080] 2. The three Lys in the B domain truncation sequence were replaced by glycine mutations, and three Lys were added to the ends of the dimer, trimer and tetramer that did not contain Lys as coupling elements. Compared with the truncated form, the amount of immunoglobulin adsorbed by the ligand after glycine substitution was increased in affinity media with different ligand coupling amounts.

[0081] 3. The three Lys in the B domain truncation sequence were replaced by arginine, and three Lys were added to the ends of the dimer, trimer and tetramer that did not contain Lys as coupling elements. Compared with the truncated form, the amount of immunoglobulin adsorbed by the ligand after arginine substitution was increased in affinity media with different ligand coupling amounts.

[0082] 4. When the ligand coupling amount is less than 15 mg / g, the performance of the ligand after arginine substitution mutation is better than that of the glycine substitution mutation group and the truncation group. The affinity medium per unit weight can bind a larger amount of immunoglobulin, thereby improving the binding capacity of immunoglobulin.

[0083] At present, the best implementation scheme of the immunoglobulin binding activity of the present invention is a modified protein, first truncating the amino acid sequence of positions 49-58 in the B domain of protein A to obtain a truncate, then performing substitution mutations on the three Lys at positions 4, 7 and 35 in the truncate, and expressing multiple truncated polypeptides after substitution mutations in series, and adding lysine as a coupling site at the end of the multimeric protein. As a ligand of an affinity medium, the protein after the modification described in this patent has the activity of binding to immunoglobulins, and it is found that compared with the truncated protein without Lys mutations at positions 4, 7 and 35, the activity of binding to immunoglobulins is significantly improved. In addition, the modified protein A maintains a good orientation when fixed on a solid phase carrier by Lys residues. In other words, the modified protein of the present invention has very useful structural and functional characteristics as an affinity ligand for immunoglobulins.

[0084] It should be noted that the simultaneous introduction of the above changes into the Z domain is also within the protection scope 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 in the protection scope of the present invention.

Claims

1. A polypeptide, characterized in that: The polypeptide comprises the amino acid sequence shown in SEQ ID NO: 2 after the amino acid sequence of positions 49 to 58 of the B domain polypeptide of protein A shown in SEQ ID NO: 1 has been truncated.

2. A polypeptide according to claim 1, characterized in that: The amino acid sequence shown in SEQ ID NO: 2 has substitution mutations at the lysine residues at positions 4, 7 and 35.

3. A polypeptide according to claim 2, characterized in that: The lysine residues at positions 4, 7 and 35 are substituted with one or more of glycine, phenylalanine, asparagine, valine, histidine, alanine, aspartic acid, leucine, threonine, tyrosine, serine and arginine.

4. A polypeptide according to claim 3, characterized in that: The three lysine residues at positions 4, 7 and 35 are all substituted by glycine, and the amino acid sequence is shown in SEQ ID NO:

3.

5. A polypeptide according to claim 3, characterized in that: The three lysine residues at positions 4, 7 and 35 are all substituted by arginine, and the amino acid sequence is shown in SEQ ID NO:

4.

6. A polymer, characterized in that: The multimer comprises 2-6 linked polypeptides, and the polypeptide is the polypeptide according to any one of claims 1-5.

7. A polymer according to claim 6, characterized in that: The polypeptides are connected directly via peptide bonds at the C-terminus and N-terminus or via a linker.

8. A polymer according to claim 6, characterized in that: The amino acid sequence of the polymer is modified by adding 1 to 10 lysine residues to the terminal.

9. A polymer according to claim 6, characterized in that: The amino acid sequences of the polymer are shown in SEQ ID NOs: 5-7, SEQ ID NOs: 15-17 and SEQ ID NOs: 25-27.

10. Use of a multimer as claimed in any one of claims 6 to 9 in an immunoglobulin affinity ligand.

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