Polypeptides, fusion multimeric proteins, and uses thereof

Abstract

The present invention relates to the technical field of biological functions of proteins, specifically to a polypeptide, a fusion multimeric protein, and uses thereof, selected from (1) a polypeptide having a substitution mutation at at least one position selected from positions 16, 25, 29, 49, and 58, compared to the native C domain of Protein A shown in SEQ ID NO.1, where position 16 is mutated by substitution to leucine or valine, position 25 is mutated by substitution to lysine, arginine, histidine, or tryptophan, position 29 is mutated by substitution to alanine, leucine, or threonine, position 49 is mutated by substitution to arginine or histidine, and position 58 is mutated by substitution to glycine, isoleucine, or alanine, and (2) a polypeptide having at least 80% or more homology with the polypeptide of (1) and carrying a substitution mutation at at least one position selected from positions 16, 25, 29, 49, and 58. The polypeptide has high alkali resistance and high loading capacity.

Description

Technical Field

[0001] This application claims the priority of a Chinese patent application filed with the China National Intellectual Property Administration on September 1, 2022, with the application number 202211064221.6 and the invention title "Polypeptide, Fusion Multimer Protein and Their Uses", the entire content of which is incorporated herein by reference.

[0002] This application relates to the technical field of the biological functions of proteins, and specifically relates to polypeptides, fusion multimer proteins and their uses.

Background Art

[0003] By February 2022, the FDA had approved a total of 109 antibody drugs. The global antibody drug market has maintained a growth rate of over 10% for eight consecutive years, exceeding $200 billion for the first time in 2021, a 16.5% increase from 2020. The isolation of antibodies is extremely important in the production of antibody drugs. Protein A affinity purification is a crucial step in the isolation and purification of antibodies. Currently, Protein A fillers are sensitive to alkaline conditions, and the elution conditions are too harsh. As the market scale of antibody purification expands, the requirements for the loading capacity and alkali resistance of Protein A fillers are becoming increasingly high. Staphylococcal Protein A (SPA) is abbreviated as Protein A and is one of the cell wall proteins of Staphylococcus aureus. In 1983, the SPA gene was cloned by Duggleby et al. and expressed in Escherichia coli (Duggleby, C.J and Jones, S.A: cloning and expression of the Staphylococcus aureus protein A gene in Escherichia coli. Nucl Acids Res. 11(1983)3065 - 3076; Lofdahl, S., Guss, B., et al; Gene for Staphylococcal protein A. Proc.Natl.Acid.Sci.USA 80(1983)697 - 701). The SPA protein molecule consists of a total of six domains, namely five highly homologous domains of E, D, A, B, and C and the X domain. The five highly homologous domains of E, D, A, B, and C on SPA all have the ability to bind to IgG and can bind to the Fc and Fab regions of most mammalian IgG (Jansson B, Uhlen M, Nygren PA. All individual domains of staphylococcal protein A show Fab binding. FEMS Immunol Med Microbiol. 1998 Jan;20(1):69 - 78.). Therefore, SPA is bound to a solid-phase carrier as an affinity ligand for antibody purification and is widely applied in the isolation and purification of antibodies.

[0004] During the antibody production process, the sample contains impurities such as lipids, nucleic acids, proteins, polysaccharides, and cell fragments, or is contaminated by virus bacteria. Therefore, these impurities may be non-specifically adsorbed onto the protein A affinity filler during the antibody purification process, contaminating the filler. Also, the protein A column requires regular in-situ cleaning (Clean In Place, CIP). To extend the service life of the protein A filler and ensure the column effect during filler use, cleaning and regeneration of the filler are necessary. The most widely used method currently is the in-situ cleaning of the chromatography column using 0.1 - 1M NaOH. However, natural protein A cannot withstand NaOH at a concentration higher than 0.1M (Garshasb, Rigi, Samira, et al. A comprehensive review on staphylococcal protein A (SpA): Its production and applications. [J]. Biotechnology & Applied Biochemistry, 2019.). With the development of genetic engineering, the alkali resistance of recombinant protein A has gradually improved. However, when manufacturing antibody drugs through industrial production, the consumption of recombinant protein A is large, its service life is limited, and the production cost increases. Therefore, protein A fillers with higher loading capacity and higher alkali resistance are an inevitable trend pursued by antibody manufacturers and also the development direction of filler manufacturers.

[0005] Antibodies generally consist of Fab and Fc domains. The interaction between the antibody Fab region and SPA significantly affects the elution pH value. A lower pH value is required for the dissociation and elution of IgG. The B-domain mutant of SPA molecule with glycine at position 29 substituted by alanine hardly interacts with the Fab region. When asparagine at position 23 of the B domain is substituted by threonine, the alkali resistance of the B-domain mutant is significantly improved (Amritkar V, Adat S, Tejwani V, et al. Engineering Staphylococcal Protein A for high-throughput affinity purification of monoclonal2antibodies[J]. Biotechnology advances, 2020:107632.). However, the modified SPA molecules still cannot meet the requirements of high loading and high alkali resistance of the current SPA molecules.

Summary of the Invention

Problems to be Solved by the Invention

[0006] Therefore, the technical problem to be solved by the present application is to provide polypeptides, fusion multimeric proteins and their uses, and the obtained fusion multimeric proteins have a higher loading capacity and higher alkali resistance.

Means for Solving the Problems

[0007] In view of this, the present application provides the following technical solutions. According to one aspect of the present application, (1) having a substitution mutation at at least one position selected from positions 16, 25, 29, 49 and 58 compared to the native C domain of Protein A shown in SEQ ID NO.1, position 16 is mutated by substitution to leucine or valine, position 25 is mutated by substitution to lysine, arginine, histidine, or tryptophan, The 29th position is mutated by substitution with alanine, leucine, or threonine, The 49th position is mutated by substitution with arginine or histidine, The 58th position is mutated by substitution with glycine, isoleucine, or alanine, (2) A polypeptide is provided that has at least 80% homology with the polypeptide of (1) and retains a substitution mutation at at least one position among the 16th, 25th, 29th, 49th, and 58th positions.

[0008] Optionally, the 16th position is mutated by substitution with leucine, and / or the 25th position is mutated by substitution with lysine, and / or the 29th is mutated by substitution with alanine, and / or the 49th position is mutated by substitution with arginine, and / or the 58th position is mutated by substitution with glycine.

[0009] Optionally, the polypeptide has the substitution mutation at the 58th position, has the substitution mutation at at least one position selected from the 16th, 25th, 29th, and 49th positions, or the polypeptide has the substitution mutation at the 49th position, has the substitution mutation at at least one position selected from the 16th, 25th, 29th, and 58th positions, or the polypeptide has the substitution mutation at the 29th position, has the substitution mutation at at least one position selected from the 16th, 25th, 49th, and 58th positions, or the polypeptide has the substitution mutation at the 25th position, has the substitution mutation at at least one position selected from the 16th, 29th, 49th, and 58th positions, or the polypeptide has the substitution mutation at the 16th position, has the substitution mutation at at least one position selected from the 25th, 29th, 49th, and 58th positions, Optionally, the polypeptide has substitution mutations at positions 16, 25, 29, 49 and 58, or Optionally, the polypeptide has substitution mutations at positions 16, 25, 49 and 58, or Optionally, the polypeptide has substitution mutations at positions 49 and 58, or Optionally, the polypeptide has substitution mutations at positions 29, 49 and 58, or Optionally, the polypeptide has substitution mutations at positions 16, 25 and 29, or Optionally, the polypeptide has substitution mutations at positions 16 and 25, or Optionally, the polypeptide has a substitution mutation at position 49, or Optionally, the polypeptide has a substitution mutation at position 25.

[0010] According to another aspect of the present application, there is provided a fusion multimeric protein comprising a fusion multimeric protein formed by fusing polypeptides.

[0011] The fusion multimeric protein further comprises polypeptide B and / or polypeptide Zmb, The polypeptide B is a. one having the amino acid sequence shown in SEQ ID NO.2, b. one selected from those having an amino acid sequence having at least 80% homology with the amino acid sequence shown in SEQ ID NO.2 of a, The polypeptide Zmb is c. one in which the amino acid sequence ML is inserted at at least one position of the amino acid sequence shown in SEQ ID NO.2 of a, and the amino acid sequence of the amino acid sequence ML is the one shown in SEQ ID NO.3, d. one selected from those in which the amino acid sequence ML is inserted at at least one position of the amino acid sequence of b, Optionally, the amino acid sequence of the polypeptide Zmb is shown in SEQ ID NO.4.

[0012] The fusion multimeric protein designates the polypeptide as polypeptide Cm, the fusion multimeric protein contains at least one of the polypeptide Cm and / or at least one of the polypeptide Zmb, Optionally, the fusion multimeric protein contains 1 to 6 of the polypeptide Cm and / or 1 to 4 of the polypeptide Zmb, Optionally, the fusion multimeric protein contains 4 of the polypeptide Cm and / or 2 of the polypeptide Zmb.

[0013] The fusion multimeric protein A. having the amino acid sequence shown in SEQ ID NO.5, B. further includes at least one functional polypeptide FLD selected from those having an amino acid sequence with at least 80% or more homology with the amino acid sequence shown in SEQ ID NO.5 of A, Optionally, the functional polypeptide FLD is located between the polypeptide Cm and the polypeptide Zmb, Optionally, the fusion multimeric protein contains, from the N-terminus to the C-terminus, 4 polypeptide Cm, 1 functional polypeptide FLD, and 2 polypeptide Zmb.

[0014] According to another aspect of the present application, 1) a nucleic acid molecule, optionally DNA or RNA, encoding the polypeptide or the fusion multimeric protein, 2) an expression cassette, recombinant vector, recombinant microorganism or transgenic cell line expressing the polypeptide or the fusion multimeric protein, 3) an expression cassette, recombinant vector, recombinant microorganism or transgenic cell line containing the nucleic acid molecule of 1), 4) a recombinant vector, recombinant microorganism or transgenic cell line containing the expression cassette of 2) or 3), 5)2) or 3) or 4) including any one of the host cells containing the recombinant vector, optionally, the recombinant vector is constructed from the E. coli expression vector pET-30a(+), optionally, the host cell is E. coli, optionally, the E. coli is E. coli BL21(DE3), to provide a biological material.

[0015] Use of the polypeptide or the fusion multimeric protein in antibody detection, isolation or purification.

[0016] According to another aspect of the present application, there is provided an affinity chromatography medium including the fusion multimeric protein and an affinity chromatography medium carrier, optionally, the affinity chromatography medium carrier includes agarose gel, dextran, cellulose, a polymer having a hydroxyl group or silica gel.

Advantages of the Invention

[0017] The technical solution of the present application has the following advantages.

[0018] 1. This application provides a polypeptide selected from: (1) having a substitution mutation at at least one position selected from positions 16, 25, 29, 49, and 58 compared to the native C domain of Protein A shown in SEQ ID NO.1, wherein position 16 is mutated by substitution to leucine or valine, position 25 is mutated by substitution to lysine, arginine, histidine, or tryptophan, position 29 is mutated by substitution to alanine, leucine, or threonine, position 49 is mutated by substitution to arginine or histidine, and position 58 is mutated by substitution to glycine, isoleucine, or alanine; (2) having at least 80% homology with the polypeptide of (1) and retaining the substitution mutation at at least one of positions 16, 25, 29, 49, and 58. Due to the high alkali resistance and high loading capacity of the polypeptide, the fusion multimeric protein produced using this polypeptide will have a higher loading capacity and higher alkali resistance. 2. The fusion multimeric protein according to this application includes a fusion multimeric protein formed by the fusion of the polypeptide, and has a higher loading capacity and higher alkali resistance. 3. The fusion multimeric protein according to this application further includes a polypeptide Zmb whose amino acid sequence is shown in SEQ ID NO.4. From the experimental results, when a filler is produced with the polypeptide Zmb domain and used for antibody purification, it is shown that the pH value of the elution buffer rises from 3.3 to 4.3. 4. The fusion multimeric protein according to this application further includes at least one functional polypeptide FLD. The insertion of the FLD reduces the steric hindrance effect of the three-dimensional structure of the fusion multimeric protein, thereby enabling the fusion multimeric protein to recombine with IgG and increasing the loading capacity of the entire fusion multimeric protein. 5. The affinity chromatography medium according to the present application includes the fusion multimeric protein and the affinity chromatography medium carrier, and the fusion multimeric protein is bound to the affinity chromatography medium carrier to serve as an affinity chromatography medium for the detection, isolation, or purification of antibodies.

[0019] To more clearly explain the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used for the description of the specific embodiments or the prior art will be briefly described below. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without creative effort.

Brief Description of the Drawings

[0020]

Figure 1

Figure 2

Figure 3

Figure 4

Modes for Carrying Out the Invention

[0021] The following examples are provided to better understand the present application. However, the present application is not limited to the best embodiment described above, and these examples do not limit the content and protection scope of the present application. Any product identical or similar to the present application as a result of being suggested by the present application or by combining the features of the present application with other prior art is included in the patent scope of the present application.

[0022] If specific experimental steps or conditions are not described in the examples, they can be implemented according to the operations or conditions of the conventional experimental steps described in the literature of the relevant field. If the reagents or instruments used are not specified by the manufacturer, they are all ordinary reagent products that can be purchased on the market.

[0023] The coding genes of the polypeptides and fusion multimeric proteins in the following examples were synthesized by Nanjing Jinrui Biotechnology Co., Ltd.

[0024] In the present application, amino acid residues may also be abbreviations of lysine (K), glycine (G), alanine (A), arginine (R), glutamic acid (E), isoleucine (I), leucine (L).

[0025] Example 1 Polypeptide Cm 1. In order to explore the alkaline resistance scheme of polypeptide Cm, in this example, specifically, a series of polypeptide Cm as follows was designed. Polypeptide C (natural): The amino acid sequence is shown in SEQ ID NO.1. Polypeptide Cm (K58G): Compared with the natural C domain of protein A shown in SEQ ID NO.1, only the lysine (K) at position 58 was mutated by substitution with glycine (G). Polypeptide Cm (K49R): Compared with the natural C domain of protein A shown in SEQ ID NO.1, only the lysine (K) at position 49 was mutated by substitution with arginine (R). Polypeptide Cm(G29A): Compared with the native C domain of protein A shown in SEQ ID NO.1, only the glycine (G) at position 29 is mutated by substitution with alanine (A). Polypeptide Cm(E25K): Compared with the native C domain of protein A shown in SEQ ID NO.1, only the glutamic acid (E) at position 25 is mutated by substitution with lysine (K). Polypeptide Cm(I16L): Compared with the native C domain of protein A shown in SEQ ID NO.1, only the isoleucine (I) at position 16 is mutated by substitution with leucine (L). Polypeptide Cm(K58G, K49R, G29A, E25K, I16L): Compared with the native C domain of protein A shown in SEQ ID NO.1, the lysine (K) at position 58 is mutated by substitution with glycine (G), the lysine (K) at position 49 is mutated by substitution with arginine (R), the glycine (G) at position 29 is mutated by substitution with alanine (A), the glutamic acid (E) at position 25 is mutated by substitution with lysine (K), and the isoleucine (I) at position 16 is mutated by substitution with leucine (L). Polypeptide Cm(K58G, K49R, E25K, I16L): Compared with the native C domain of protein A shown in SEQ ID NO.1, the lysine (K) at position 58 is mutated by substitution with glycine (G), the lysine (K) at position 49 is mutated by substitution with arginine (R), the glutamic acid (E) at position 25 is mutated by substitution with lysine (K), and the isoleucine (I) at position 16 is mutated by substitution with leucine (L). Polypeptide Cm(K58G, K49R): Compared with the native C domain of protein A shown in SEQ ID NO.1, the lysine (K) at position 58 is mutated by substitution with glycine (G), 49 lysines (K) were mutated by substitution with arginines (R). Polypeptide Cm (K58G, K49R, G29A): Compared to the native C domain of protein A shown in SEQ ID NO.1, the lysine (K) at position 58 was mutated by substitution with glycine (G), the lysine (K) at position 49 was mutated by substitution with arginine (R), and the glycine (G) at position 29 was mutated by substitution with alanine (A). Polypeptide Cm (G29A, E25K, I16L): Compared to the native C domain of protein A shown in SEQ ID NO.1, the glycine (G) at position 29 was mutated by substitution with alanine (A), the glutamic acid (E) at position 25 was mutated by substitution with lysine (K), and the isoleucine (I) at position 16 was mutated by substitution with leucine (L). Polypeptide Cm (E25K, I16L): Compared to the native C domain of protein A shown in SEQ ID NO.1, the glutamic acid (E) at position 25 was mutated by substitution with lysine (K), and the isoleucine (I) at position 16 was mutated by substitution with leucine (L). Furthermore, the substitution mutation at position 16 in the above polypeptide Cm may also be related to valine. The substitution mutation at position 25 may also be related to arginine, histidine, or tryptophan. The substitution mutation at position 29 may also be related to leucine or threonine. The substitution mutation at position 49 may also be related to histidine. The substitution mutation at position 58 may also be related to isoleucine or alanine.

[0026] Example 2 In this example, the following polypeptide Zmb was designed. Polypeptide B: The amino acid sequence is shown in SEQ ID NO.2. Polypeptide Zmb: The amino acid sequence ML shown in SEQ ID NO.3 is inserted between the 20th and 21st amino acids of the amino acid sequence shown in SEQ ID NO.2. That is, the amino acid sequence of polypeptide Zmb is shown in SEQ ID NO.4.

[0027] Example 3 In this example, a functional polypeptide FLD with the amino acid sequence shown in SEQ ID NO.5 was designed.

[0028] Example 4 Fusion-type multimeric protein In this example, using the polypeptides of Examples 1 to 3, a series of fusion-type multimeric proteins were designed as follows. CC: It is an amino acid sequence in which six polypeptides C (natural) are fused in order from the N-terminus to the C-terminus, and the sequence is C-C-C-C-C-C. CmCm(K58G): It is an amino acid sequence in which six polypeptides Cm(K58G) are fused in order from the N-terminus to the C-terminus. CmCm(K49R): It is an amino acid sequence in which six polypeptides Cm(K49R) are fused in order from the N-terminus to the C-terminus. CmCm(G29A): It is an amino acid sequence in which six polypeptides Cm(G29A) are fused in order from the N-terminus to the C-terminus. CmCm(E25K): It is an amino acid sequence in which six polypeptides Cm(E25K) are fused in order from the N-terminus to the C-terminus. CmCm(I16L): It is an amino acid sequence in which six polypeptides Cm(I16L) are fused in order from the N-terminus to the C-terminus. CmCm(K58G, K49R, G29A, E25K, I16L): It is an amino acid sequence in which six polypeptides Cm(K58G, K49R, G29A, E25K, I16L) are fused in order from the N-terminus to the C-terminus. CmCm(K58G, K49R, E25K, I16L): It is an amino acid sequence in which six polypeptides Cm(K58G, K49R, E25K, I16L) are fused in order from the N-terminus to the C-terminus. CmCm(K58G, K49R): It is an amino acid sequence in which six polypeptides Cm(K58G, K49R) are fused in order from the N-terminus to the C-terminus. CmCm(K58G, K49R, G29A): It is an amino acid sequence in which six polypeptides Cm(K58G, K49R, G29A) are fused in order from the N-terminus to the C-terminus. CmCm(G29A, E25K, I16L): It is an amino acid sequence in which six polypeptides Cm(G29A, E25K, I16L) are fused in order from the N-terminus to the C-terminus. CmCm(E25K, I16L): It is an amino acid sequence in which six polypeptides Cm(E25K, I16L) are fused in order from the N-terminus to the C-terminus. CDB: It is an amino acid sequence in which four polypeptides C (natural), one functional polypeptide FLD, and two polypeptides B are fused from the N-terminus to the C-terminus, and the sequence is C-C-C-C-FLD-B-B. CB: It is an amino acid sequence in which four polypeptides C (natural) and two polypeptides B are fused from the N-terminus to the C-terminus, and the sequence is C-C-C-B-B. CmZmb: It is an amino acid sequence in which four polypeptides Cm, one functional polypeptide FLD, and two polypeptides Zmb are fused in order from the N-terminus to the C-terminus, and the sequence is Cm-Cm-Cm-Cm-FLD-Zmb-Zmb. Its amino acid sequence is shown in SEQ ID NO.6, and its gene sequence is shown in SEQ ID NO.7. Furthermore, the polypeptide Cm in the above fusion multimer protein is involved in The substitution mutation at position 16 may be related to valine. The substitution mutation at position 25 may be related to arginine, histidine or tryptophan. The substitution mutation at position 29 may be related to leucine or threonine. The 49 - position substitution mutation may relate to histidine. The 58 - position substitution mutation may relate to isoleucine or alanine.

[0029] Example 5 Production of Fusion Multimeric Protein In this example, the fusion multimeric protein designed in Example 4 was produced according to the following process.

[0030] (1) Construction of Recombinant Vector The coding gene of the fusion multimeric protein and the expression vector pET - 30a(+) were digested with a double enzyme of NdeI (purchased from Sangon Biotech (Shanghai) Co., Ltd., B600120) and HindIII (purchased from Sangon Biotech (Shanghai) Co., Ltd., B600184). After recovering the corresponding fragments, they were ligated (DNA Gel Recovery Kit, purchased from Shanghai Biyuntian Biotechnology Co., Ltd., D0056). The ligation product and E. coli TOP10 competent cells were uniformly mixed, left standing on ice for 30 min, heat - shocked at 42 °C for 90 s in a water bath, left standing on ice for 3 min, and transformation was carried out. 100 μl of room - temperature LB liquid medium was added, and shaking culture was carried out at 37 °C and 220 revolutions per minute for 60 minutes. The bacterial solution was uniformly mixed and spread on a kanamycin (purchased from Sangon Biotech (Shanghai) Co., Ltd., A506636) - resistant plate. This plate was inverted and cultured overnight at 37 °C to screen for transformants with kanamycin resistance, and a recombinant vector expressing the fusion multimeric protein was screened. For example, the recombinant vector expressing the screened fusion multimeric protein CmZmb was named pET - 30a - CmZmb, and by analogy, the recombinant vectors of other fusion multimeric proteins were named.

[0031] (2) Construction of Recombinant Bacteria The fresh BL21(DE3) bacterial solution was treated in an ice bath for 30 min, centrifuged at 4°C and 1000 g for 10 min. The precipitate was resuspended in 0.1 mol / L MgCl2-CaCl2 (80 mmol MgCl2 and 80 mmol CaCl2, MgCl2 and CaCl2 were purchased from Sinopharm Chemical Reagent Co., Ltd., analytical pure, and the remaining chemical reagents had the same purity), centrifuged at 4°C and 1000 g for 10 min, and then resuspended in 0.1 mol / L CaCl2 solution to prepare BL21(DE3) competent cells. The recombinant vector (such as pET-30a-CmZmb) screened in step (1) was transformed into Escherichia coli BL21(DE3) competent cells, and the transformants were screened on a kanamycin-containing plate. As a result of sequencing, the correct recombinant transformant was confirmed and named BL21-pET30a-CmZmb. By analogy, the recombinant transformants of other fusion multimeric proteins were named.

[0032] (3) Fermentation of recombinant bacteria As a result of sequencing, the correct recombinant transformant (such as BL21-pET30a-CmZmb) was inoculated into LB medium at an inoculation amount of 0.5% (volume %), cultured overnight at 37°C until the OD 600 reached 0.8, induced by adding lactose with a final concentration of 10 g / L, centrifuged 3 - 5 hours later, and the bacterial solution was collected.

[0033] 40 μl of the bacterial solution (the bacterial solution before lactose induction and the bacterial solution after lactose induction respectively) was added with 10 μl of 5× injection buffer (provided by Shaanxi Proanthocyanidin Biotechnology Development Co., Ltd., product number: 10137-1), boiled in boiling water for 5 min, and 20 μl was injected for SDS-PAGE gel electrophoresis. As a result, as shown in Figure 1, in the lactose-induced BL21-pET30a-CmZmb bacterial solution, a new protein band appeared at the position of 44 kD (see lane 2 in Figure 1). In the non-lactose-induced BL21-pET30a-CmZmb bacterial solution, such a band did not appear or the color of this band was faint (see lane 1 in Figure 1). From this, it was shown that the fusion multimeric protein CmZmb was induced to be expressed in BL21-pET30a-CmZmb. Similarly, for other fusion multimeric proteins in Example 4, the expression was successfully induced.

[0034] (4) Purification Sodium chloride for analysis was added to a solution containing 20 mM PB (prepared using an aqueous solution of disodium hydrogen phosphate dodecahydrate at 5.8018 g / L and an aqueous solution of disodium hydrogen phosphate dihydrate at 0.5928 g / L, and the buffer solution was prepared by ordinary methods) to a concentration of 11.688 g / L, and the pH was adjusted to pH 7.4 with an acid or alkali aqueous solution to obtain Buffer A. The bacterial solution collected in step (3) above was suspended in Buffer A at a weight ratio of 1:10, with the decomposition times set to 20 min, 30 min, and 40 min respectively, sonicated, centrifuged at room temperature, and then the supernatant was collected to obtain decomposition solution supernatants 1, 2, and 3. At the same time, the supernatant of the lysate 2 was subjected to nickel column affinity purification (as in the following lysate supernatant 2 (lysis time 30 min), nickel column affinity purification was performed until 40% of solution B eluted, the eluate was collected, and the resulting eluate was divided into two to obtain eluate 1 and eluate 2). As shown in the results of SDS-PAGE electrophoresis detection in lanes 3 - 7 of Figure 1 (there was no difference in the results of the samples using three types of decomposition times. As in lanes 3 - 5 of Figure 1, the decomposition solution supernatant 1 in lane 3 corresponded to a decomposition time of 20 min, the decomposition solution supernatant 2 in lane 4 corresponded to a decomposition time of 30 min, and the decomposition solution supernatant 3 in lane 5 corresponded to a decomposition time of 40 min. As in lanes 6 - 7 of Figure 1, there was no obvious difference in the results of eluate 1 and eluate 2), most of the fusion-type multimeric protein was expressed in the Escherichia coli periplasm, and the expression level in the precipitate was relatively low.

[0035] The supernatant of the lysate 2 (lysis time: 30 min) was subjected to nickel column affinity purification, and the specific purification steps are shown below. The flow rate of the purification apparatus (AKTA, Purifier) was adjusted to 3 ml / min, and the column was equilibrated with Buffer A. The baselines of A280 nm (detection wavelength), A231 nm (detection wavelength), and A215 nm (detection wavelength) were stabilized for about 10 column volumes (CV), and the baseline was set to zero. The sample (60 - 120 ml of the lysate supernatant) was injected with the A pump. The column was rinsed with Buffer A until the baseline was stabilized for about 10 CV. Impurities were washed with 8% (v / v) Buffer B (Buffer B was prepared by adding imidazole with a final concentration of 17.02 g / L to Buffer A; imidazole was purchased from Sinopharm Chemical Reagent Co., Ltd.) until the baseline was stabilized for about 10 CV. The target protein was eluted with 40% (v / v) Buffer B until the baseline was stabilized for about 10 CV. Elution was performed with Buffer B (100% (v / v)) for about 3 CV to stabilize the baseline. (This protein has a low absorption at A280 nm and a high absorption at A215 nm, but imidazole has a great influence on A215 nm. When collecting the protein, it is necessary to repeatedly confirm the collection range. When the concentration is low, collection is started at the beginning of the peak of A215 nm or A231 nm, and when the concentration is high, collection is started at the beginning of the peak of A280 nm.) The column and the chromatography system were washed with Buffer A until the baseline was stabilized for about 10 CV. The purified recombinant protein with characteristic peaks was collected. For example, the purity of the fusion multimeric protein CmZmb was 80% or higher (see the lanes in Figure 2 for the results). Similarly, the purity of other fusion multimeric proteins was also 80% or higher.

[0036] Example 6 Preparation of Affinity Chromatography Medium In this example, an affinity chromatography medium was prepared as follows.

[0037] Using the fusion multimeric protein obtained in Example 5, affinity chromatography media were each prepared. The specific steps are shown below. To 100 g of agarose gel 4FF (Xi'an Blue Dawn New Materials Co., Ltd.), 2 to 30 mL (15 mL in this example) of cyanogen bromide was added, and the reaction was carried out at -10°C to 10°C (0°C in this example) for 15 to 30 min (22 min in this example). Then, 2 to 50 mL (25 mL in this example) of triethylamine was added, and the reaction was carried out at -10°C to 10°C (0°C in this example) for 15 to 60 min (35 min in this example), followed by washing with acetone. The fusion multimeric protein was dissolved in 0.2 to 2 g (1 g in this example) of pure water, added to the above agarose gel 4FF, adjusted to pH = 7 to 11 (pH = 8 in this example), and reacted overnight at 0°C to 20°C (10°C in this example). Then, 0.1 to 10% (volume%, 5% in this example) of an ethanolamine solution with pH = 7 to 11 was added, and the reaction was carried out overnight at 0°C to 40°C (20°C in this example) to obtain an affinity chromatography medium. The prepared affinity chromatography medium was stored in a 20% (volume%) ethanol solution.

[0038] Experimental Example 1 Dynamic Loading Capacity Test of Fusion Multimeric Protein Filler Programming and experiments were carried out using an AKTA pure chromatography system. The column medium was the affinity chromatography medium prepared in Example 6. 1.1 ml of the affinity chromatography medium was uniformly mixed with 2 ml of a 20% (v / v) ethanol aqueous solution, and then loaded into the column at a flow rate of 8 rpm / min using a constant flow pump (purchased from Shanghai Qingpu Luxi Instrument Factory). The detection wavelength was set at 280 nm, the pre-column pressure was set at 0.3 MPa, the column was connected to the system, the flow rate was set at 1.0 ml / min, and elution was performed with 10 CV of mobile phase B1, followed by equilibration with 15 CV of mobile phase A1. A sample (2 mg / ml IgG, purchased from Beijing Solarbio Science & Technology Co., Ltd., SP031) was injected at a flow rate of 0.2 ml / min, and the absorption value at the time of injection up to 280 nm was 90 mAu. Mobile phase B1 was set as the constant elution mobile phase at 100% (v / v), the flow rate was set at 1.0 ml / min, and elution was performed until the baseline was stable, and the eluate was collected. Finally, regeneration was performed with 10 ml of mobile phase A1 at a flow rate of 1.0 ml / min. The absorption value was dynamically detected from the start to the end of the program.

[0039] Mobile phase A1: A mixed solution containing 20 mM PB and 0.15 M NaCl, pH 7.4 Mobile phase B1: 0.1 M citric acid - sodium citrate buffer, pH 3.3 Dynamic loading capacity = (V A × C0) / V C (mg / ml gel) C0 is the concentration of the target protein in the sample, V A is the total injection volume when the concentration of the target sample in the breakthrough curve reaches 5% C0, and V C is the total column bed volume (see Liu Ying, Zeng Jianzhu, Yu ▲Biao▼ et al. Method for measuring the dynamic loading capacity of an affinity chromatography packing: Refer to the specification of Chinese Patent Application Publication No. 114280208). As a result of detecting the dynamic loading capacity of fillers produced with CmZmb, CC, CDB, and CB (polypeptide C in CC, CDB, and CB is all polypeptide C (natural)), as shown in Figure 3, the dynamic loading capacity of the filler produced with CmZmb was higher than that of the fillers produced with CC, CDB, and CB.

[0040] As a result of implementing by the above method, when the elution buffer was pH 4.3 (that is, mobile phase B1 was pH 4.3), the elution efficiencies of CmZmb, CC, CDB, and CB were 86.6%, 56.8%, 87.9%, and 62.3% respectively. The affinity chromatography medium produced with CmZmb had a higher efficiency when eluting the target protein with a pH 4.3 buffer compared to other affinity chromatography media.

[0041] Experimental Example 2 Alkaline Resistance Test of Fusion Multimer Protein Filler The experiment was carried out by programming using an AKTA pure chromatography system. The column medium was the affinity chromatography medium produced in Example 6. 1.1 ml of the affinity chromatography medium was uniformly mixed with 2 ml of a 20% (volume%) aqueous ethanol solution, and then put into the column at a flow rate of 8 rpm / min using a constant flow pump (purchased from Shanghai Qingpu Luxi Instrument Factory). The detection wavelength was set to 280, the pre-column pressure of the column was set to 0.3 MPa, the column was connected to the system, and it was equilibrated for 10 min at a flow rate of 1 mL / min using buffer A2 to wash away the ethanol in the column. After washing the column with 0.5 M NaOH at a flow rate of 0.2 mL / min for 15 min, the column was washed with buffer A2 at a flow rate of 0.5 mL / min for 10 min. This was repeated 100 cycles, and the dynamic loading capacity was measured every 10 cycles (for the calculation method of the dynamic loading capacity, refer to Experimental Example 1). Buffer A2: A mixed solution containing 20 mM PB and 0.15 M NaCl, pH 7.4.

[0042] Refer to Figure 4 for a part of the results of the alkaline resistance test when producing a fusion multimer protein with only polypeptide C (natural) or polypeptide Cm.

[0043]

Table 1

[0044] 2. The results of the alkali resistance test of the fillers manufactured with CmZmb, CC, CDB, and CB are shown in FIG. 4. From this figure, it can be seen that the dynamic loading percentage in the alkali resistance test of the filler manufactured with CmZmb was higher than that of CC, CDB, and CB.

[0045] Obviously, the above embodiments are merely examples for clear explanation and do not limit the embodiments. For those skilled in the art, various forms of changes and modifications can be made based on the above description. Here, it is not necessary to cover all embodiments, nor is it possible to cover them all. The obvious changes and modifications derived therefrom are still within the protection scope of the present invention and creation.

Claims

1. A fusion multimeric protein formed by the fusion of polypeptides, wherein the fusion multimeric protein comprises at least one polypeptide Cm, wherein the polypeptide Cm has the natural C domain of Protein A shown in SEQ ID NO. 1 mutated by substitution of leucine at position 16, mutated by substitution of lysine at position 25, mutated by substitution of alanine at position 29, mutated by substitution of arginine at position 49, and contains an amino acid sequence mutated by substitution of glycine at position 58, characterized in that it is a fusion multimeric protein.

2. further comprises polypeptide B and / or polypeptide Zmb, wherein the amino acid sequence of the polypeptide B is the amino acid sequence shown in SEQ ID NO. 2, wherein the amino acid sequence of the polypeptide Zmb is the amino acid sequence shown in SEQ ID NO. 4, characterized in that it is the fusion multimeric protein according to Claim 1.

3. the fusion multimeric protein comprises 1 to 6 polypeptides Cm and / or 1 to 4 polypeptides Zmb, or the fusion multimeric protein comprises 4 polypeptides Cm and / or 2 polypeptides Zmb, characterized in that it is the fusion multimeric protein according to Claim 2.

4. further comprises at least one functional polypeptide FLD, wherein the amino acid sequence of the functional polypeptide FLD is the amino acid sequence shown in SEQ ID NO. 5, characterized in that it is the fusion multimeric protein according to Claim 1.

5. the functional polypeptide FLD is located between the polypeptide Cm and the polypeptide Zmb, and the amino acid sequence of the polypeptide Zmb is the amino acid sequence shown in SEQ ID NO. 4, the fusion multimeric protein according to Claim 4.

6. the fusion multimeric protein comprises 4 polypeptides Cm, 1 functional polypeptide FLD, and 2 polypeptides Zmb from the N-terminus to the C-terminus, and the amino acid sequence of the polypeptide Zmb is the amino acid sequence shown in SEQ ID NO. 4, characterized in that it is the fusion multimeric protein according to Claim 4.

7. An affinity chromatography medium, comprising the fusion multimeric protein according to claim 1 and an affinity chromatography medium carrier.

8. The affinity chromatography medium carrier according to claim 7, characterized in that it comprises agarose gel, dextran, cellulose, a polymer having a hydroxyl group, or silica gel.

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