A method for preparing a recombinant protein a protein and an affinity chromatography medium

Abstract

The application discloses a kind of recombinant ProteinA protein and the preparation method of affinity chromatography medium, the protein is the mutant of parent immunoglobulin binding protein defined in SEQ ID NO2;The preparation method of the affinity chromatography medium includes the following steps: S1, the gene of recombinant ProteinA protein is synthesized, S2, respectively constructs expression vector using the gene synthesized in the S1, S3, is expressed and purified to obtain the recombinant ProteinA solution of sequence corresponding SEQ ID, S4, using the recombinant ProteinA preparation obtains affinity chromatography medium.Beneficial effects: the application greatly improves the chemical stability of ProteinA in lye by redesigning amino acid sequence, to provide a ProteinA with specific binding effect for antibody, it has good chemical stability under alkaline condition, the binding capacity of recombinant ProteinA affinity chromatography medium is significantly improved, can tolerate 0.5-1.0M NaOH in situ cleaning, IgG binding capacity 60-90mg / mL.

Description

[0001] This application is a divisional application of patent application filed on May 11, 2021, with application number CN202110525261.5 and invention title "A method for preparing recombinant Protein A protein and affinity chromatography medium". Technical Field

[0002] This invention relates to the field of protein engineering technology, and more specifically, to a method for preparing recombinant Protein A protein and affinity chromatography medium. Background Technology

[0003] Antibody drugs have demonstrated excellent efficacy and safety in treating diseases, and have gradually played a crucial role in the treatment of oncology, cardiovascular diseases, and especially autoimmune diseases. They are currently the fastest-growing class of biological drugs. Thanks to the construction of high-expression cell lines, improvements in culture media, and the continuous expansion of reactor scale, monoclonal antibody drug technology has developed rapidly over the past few decades. In the preparation of monoclonal antibody drugs, antibody purification processes are particularly important, with antibody capture playing a key role. Protein A affinity chromatography is currently the most widely used and technologically mature method in antibody capture and purification, with over 70% of antibody drug purification processes utilizing this technology.

[0004] However, currently used Protein A affinity chromatography media have the following drawbacks in antibody drug purification: low loading capacity; low antibody recovery rate; low pH of the eluent during elution, leading to denaturation of some antibodies and reducing recovery rate; high ligand shedding rate; and problems such as high cost, short lifespan, and demanding operating conditions. Furthermore, to reduce costs, the reuse of Protein A affinity chromatography media is essential. After antibody elution, the Protein A affinity chromatography media needs to undergo rigorously validated and standardized in-situ washing (CIP) to remove impurities such as contaminating proteins, residual antibodies and their polymers, and endotoxins. The conditions for in-situ washing (treatment with 0.5 mol / L to 1.0 mol / L sodium hydroxide solution) are very demanding for Protein A affinity chromatography media, directly resulting in the destruction of the higher-order structure of Protein A, a decrease in the media's adsorption capacity, and a reduced lifespan. In existing technologies, the stability of Protein A affinity chromatography media under alkaline conditions is not ideal, thus affecting its application. Therefore, improving the performance of Protein A affinity chromatography media has become a major technical bottleneck in the preparation of monoclonal antibody drugs and a key research area that urgently needs to be addressed. Summary of the Invention

[0005] In view of the problems in the related technologies, the present invention proposes a configuration method to overcome the above-mentioned technical problems existing in the existing related technologies.

[0006] Therefore, the specific technical solution adopted by the present invention is as follows:

[0007] According to one aspect of the present invention, a recombinant Protein A protein is provided, said recombinant Protein A protein being a mutant containing the E or D domain specified in SEQ ID NO 1 or SEQ ID NO 2.

[0008] Furthermore, in the mutant of the E domain specified in SEQ ID NO 1, the glutamine at positions 2, 8, and 42 has been mutated to an amino acid selected from tyrosine, aspartic acid, phenylalanine, and isoleucine.

[0009] In the mutant of the E domain specified in SEQ ID NO 1, the asparagine at position 16 has been mutated to an amino acid selected from threonine, serine, glutamic acid, arginine, tyrosine, leucine, and isoleucine.

[0010] In the mutant of the E domain specified in SEQ ID NO 1, the glycine at positions 22 and 39 has been mutated to an amino acid selected from alanine and serine.

[0011] Furthermore, in the mutant of the D domain specified in SEQ ID NO 2, the asparagine at positions 6, 9, and 26 has been mutated to an amino acid selected from threonine, serine, glutamic acid, arginine, tyrosine, leucine, and isoleucine.

[0012] In the mutant of the D domain specified in SEQ ID NO 2, the glutamine at position 12 has been mutated to an amino acid selected from tyrosine, aspartic acid, phenylalanine, and isoleucine.

[0013] In the mutant of the D domain specified in SEQ ID NO 2, the glutamic acid at positions 18 and 56 has been mutated to an amino acid selected from glycine, leucine, arginine, aspartic acid, threonine, histidine, serine, and phenylalanine.

[0014] In the mutant of the D domain specified in SEQ ID NO 2, the glycine at positions 32 and 49 has been mutated to amino acids selected from alanine and serine.

[0015] Furthermore, the mutations are selected from:

[0016] Q2Y,Q2D,Q2F,Q2I,Q8Y,Q8D,Q8F,Q8I,Q42Y,Q42D,Q42F,Q42I,N16T,N16S,N16E,N16R,N16Y,N16L,N16I,G22A,G22S,G39A,G39S; and contains the following sequence SEQ ID NO 3. SEQ ID NO4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22.

[0017] Furthermore, the mutations are selected from:

[0018] N6T, N6S, N6E, N6R, N6Y, N6L, N6I, N9T, N9S, N9E, N9R, N9Y, N9L, N9I, N26T, N26S, N26E, N26R, N26Y, N26L, N26I, Q12Y, Q12D, Q12F, Q12I, E18G, E18L, E18R, E18D, E18T, E18H, E18S, E18F, E56G, E56L, E56R, E56D, E56T, E56H, E56S, E56F, G32A, G32S, G49A, G49S; and containing the following sequences: SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27. SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41. SEQ ID NO. 42.

[0019] Furthermore, the mutant comprises 4-8 repeating units and includes the following sequences: SEQ ID NO43, SEQ ID NO44, SEQ ID NO45, SEQ ID NO46, SEQ ID NO47, SEQ ID NO48, SEQ ID NO49, SEQ ID NO50, SEQ ID NO51, and SEQ ID NO52.

[0020] Furthermore, the recombinant Protein A protein contains a coupling group of a cysteine ​​residue at its C-terminus.

[0021] According to another aspect of the present invention, a method for preparing an affinity chromatography medium is provided, wherein the ligand of the affinity chromatography medium is the recombinant Protein A protein described above, and the method for preparing the affinity chromatography medium includes the following steps:

[0022] S1, the gene that synthesizes recombinant Protein A;

[0023] S2. Construct expression vectors using the genes synthesized in S1;

[0024] S3. Culture and express the purified protein A solution corresponding to the SEQ ID;

[0025] S4. An affinity chromatography medium is prepared using the recombinant Protein A.

[0026] Furthermore, the expression vector in S2 is PET23a.

[0027] Furthermore, the process of culturing, expressing, and purifying the recombinant Protein A solution with the corresponding sequence in S3 specifically includes the following steps:

[0028] S31. A single recombinant plasmid was transformed into Escherichia coli, and then fermented and induced to express the plasmid using LB liquid medium.

[0029] S32. After fermentation, collect the cells and use thermal lysis to break the cell wall to release the expression product, then separate it by centrifugation.

[0030] S33. The separated liquid is purified by passing it through an IgG affinity medium.

[0031] The beneficial effects of this invention are as follows:

[0032] 1) This invention significantly improves the chemical stability of Protein A in alkaline solution by redesigning the amino acid sequence, thereby providing a Protein A that specifically binds to antibodies. It has good chemical stability under alkaline conditions, and the binding capacity of recombinant Protein A to affinity chromatography media is significantly improved. It can withstand in-situ washing with 0.5-1.0M NaOH, and the IgG binding capacity is 60-90 mg / ml.

[0033] 2) The present invention also provides a method for preparing affinity chromatography media using Protein A of the mutant domain as a ligand. Compared with affinity chromatography media using domain Z and its oligomers as ligands, the affinity chromatography media prepared by the present invention has superior antibody binding ability. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments 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.

[0035] Figure 1 This is a flowchart of a method for preparing affinity chromatography media according to an embodiment of the present invention;

[0036] Figure 2 This is a schematic diagram showing the alignment of the Protein A protein domains defined by SEQ ID NO 1, SEQ ID NO 2, and SEQ ID NO 53 according to an embodiment of the present invention;

[0037] Figure 3 This is a schematic diagram showing the results of the stability of the material against 0.5M NaOH alkali according to Example 1 of the present invention;

[0038] Figure 4 This is a schematic diagram showing the results of the stability of the substance to 1.0M NaOH alkali according to Example 2 of the present invention. Detailed Implementation

[0039] To further illustrate the various embodiments, the present invention provides accompanying drawings, which are part of the disclosure of the present invention. These drawings are mainly used to illustrate the embodiments and can be used in conjunction with the relevant descriptions in the specification to explain the operating principles of the embodiments. With reference to these drawings, those skilled in the art should be able to understand other possible implementation methods and the advantages of the present invention. The components in the drawings are not drawn to scale, and similar component symbols are generally used to represent similar components.

[0040] According to embodiments of the present invention, a method for preparing recombinant Protein A protein and affinity chromatography medium is provided.

[0041] According to one aspect of the present invention, a recombinant ProteinA protein is provided, which is a mutant of the parental immunoglobulin-binding protein as defined in SEQ ID NO 1 and SEQ ID NO 2.

[0042] Protein A's protein domains are mainly composed of mutants of E, D, A, B, C, or domains Z and Zvar, among which SEQ ID NO 1 (E domain), SEQ ID NO 2 (D domain), and SEQ ID NO 53 (Zvar) are shown in [reference needed]. Figure 2 As shown. The protected object in this embodiment consists of mutants of SEQ ID NO 1 (D domain) and SEQ ID NO 2 (E domain), highlighting its significant effects in terms of alkali resistance and binding capacity.

[0043] Specifically, the recombinant Protein A protein is a mutant containing the E or D domain specified in SEQ ID NO 1 or SEQ ID NO 2.

[0044] In the mutant of the E domain specified in SEQ ID NO 1, the glutamine at positions 2, 8, and 42 has been mutated to amino acids selected from tyrosine, aspartic acid, phenylalanine, and isoleucine.

[0045] In the mutant of the E domain specified in SEQ ID NO 1, the asparagine at position 16 has been mutated to an amino acid selected from threonine, serine, glutamic acid, arginine, tyrosine, leucine, and isoleucine.

[0046] In the mutant of the E domain specified in SEQ ID NO 1, the glycine at positions 22 and 39 has been mutated to an amino acid selected from alanine and serine.

[0047] According to the mutant described above, the mutation is selected from:

[0048] Q2Y,Q2D,Q2F,Q2I,Q8Y,Q8D,Q8F,Q8I,Q42Y,Q42D,Q42F,Q42I,N16T,N16S,N16E,N16R,N16Y,N16L,N16I,G22A,G22S,G39A,G39S; and contains the following sequence SEQ ID NO 3. SEQ ID NO4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22.

[0049] In the mutant of the D domain specified in SEQ ID NO 2, the asparagine at positions 6, 9, and 26 has been mutated to an amino acid selected from threonine, serine, glutamic acid, arginine, tyrosine, leucine, and isoleucine.

[0050] In the mutant of the D domain specified in SEQ ID NO 2, the glutamine at position 12 has been mutated to an amino acid selected from tyrosine, aspartic acid, phenylalanine, and isoleucine.

[0051] In the mutant of the D domain specified in SEQ ID NO 2, the glutamic acid at positions 18 and 56 has been mutated to an amino acid selected from glycine, leucine, arginine, aspartic acid, threonine, histidine, serine, and phenylalanine.

[0052] In the mutant of the D domain specified in SEQ ID NO 2, the glycine at positions 32 and 49 has been mutated to amino acids selected from alanine and serine;

[0053] According to the mutant described above, the mutation is selected from:

[0054] N6T, N6S, N6E, N6R, N6Y, N6L, N6I, N9T, N9S, N9E, N9R, N9Y, N9L, N9I, N26T, N26S, N26E, N26R, N26Y, N26L, N26I, Q12Y, Q12D, Q12F, Q12I, E18G, E18L, E18R, E18D, E18T, E18H, E18S, E18F, E56G, E56L, E56R, E56D, E56T, E56H, E56S, E56F, G32A, G32S, G49A, G49S; and containing the following sequences: SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27. SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41. SEQ ID NO. 42.

[0055] In one embodiment, the mutant comprises 4-8 repeating units and includes the following sequences: SEQ ID NO43, SEQ ID NO44, SEQ ID NO45, SEQ ID NO46, SEQ ID NO47, SEQ ID NO48, SEQ ID NO49, SEQ ID NO50, SEQ ID NO51, and SEQ ID NO52.

[0056] In one embodiment, the recombinant Protein A protein contains a coupling group of a cysteine ​​residue at its C-terminus.

[0057] In some embodiments, the recombinant Protein A protein comprises or is substantially composed of sequences selected from the following:

[0058] SEQ ID NO 3(Q2Y)

[0059] AYQNAFYQVL NMPNLNADQR NGFIQSLKDD PSQSANVLGE AQKLNDSQAP K

[0060] SEQ ID NO 4(Q2D)

[0061] ADQNAFYQVL NMPNLNADQR NGFIQSLKDD PSQSANVLGE AQKLNDSQAP K

[0062] SEQ ID NO 5(Q2F)

[0063] AFQNAFYQVL NMPNLNADQR NGFIQSLKDD PSQSANVLGE AQKLNDSQAP K

[0064] SEQ ID NO 6(Q2I)

[0065] AIQNAFYQVL NMPNLNADQR NGFIQSLKDD PSQSANVLGE AQKLNDSQAP K

[0066] SEQ ID NO 7(Q2Y,Q8D)

[0067] AYQNAFYDVL NMPNLNADQR NGFIQSLKDD PSQSANVLGE AQKLNDSQAP K

[0068] SEQ ID NO 8(Q2Y,Q42F)

[0069] AYQNAFYQVL NMPNLNADQR NGFIQSLKDD PSQSANVLGE AFKLNDSQAP K

[0070] SEQ ID NO 9(Q2I,N16T)

[0071] AIQNAFYQVL NMPNLTADQR NGFIQSLKDD PSQSANVLGE AQKLNDSQAP K

[0072] SEQ ID NO 10(Q8F,G22A)

[0073] AQQNAFYFVL NMPNLNADQR NAFIQSLKDD PSQSANVLGE AQKLNDSQAP K

[0074] SEQ ID NO 11(Q2D,Q42F,N16S)

[0075] ADQNAFYQVL NMPNLSADQR NGFIQSLKDD PSQSANVLGE AFKLNDSQAP K

[0076] SEQ ID NO 12(Q2I,Q8F,G22A)

[0077] AIQNAFYFVL NMPNLNADQR NAFIQSLKDD PSQSANVLGE AQKLNDSQAP K

[0078] SEQ ID NO 13(Q8D,N16R,G22A)

[0079] AQQNAFYDVL NMPNLRADQR NAFIQSLKDD PSQSANVLGE AQKLNDSQAP K

[0080] SEQ ID NO 14(G22S,G39S,Q42D)

[0081] AQQNAFYQVL NMPNLNADQR NSFIQSLKDD PSQSANVLSE ADKLNDSQAP K

[0082] SEQ ID NO 15(Q8F,N16E,G39A,Q42D)

[0083] AQQNAFYFVL NMPNLEADQR NGFIQSLKDD PSQSANVLAE ADKLNDSQAP K

[0084] SEQ ID NO 16(Q2I,Q8D,Q42F,N16Y)

[0085] AQQNAFYQVL NMPNLNADQR NGFIQSLKDD PSQSANVLGE AQKLNDSQAP K

[0086] SEQ ID NO 17(Q8Y,N16L,G39A,Q42I)

[0087] AQQNAFYYVL NMPNLLADQR NGFIQSLKDD PSQSANVLAE AIKLNDSQAP K

[0088] SEQ ID NO 18(Q2I,Q8D,N16I,G39A,Q42F)

[0089] AIQNAFYDVL NMPNLIADQR NGFIQSLKDD PSQSANVLAE AFKLNDSQAP K

[0090] SEQ ID NO 19(Q2D,Q8F,N16T,G22A,G39S)

[0091] ADQNAFYFVL NMPNLTADQR NAFIQSLKDD PSQSANVLSE AQKLNDSQAP K

[0092] SEQ ID NO 20(Q8Y,N16L,G22A,G39S,Q42I)

[0093] AQQNAFYYVL NMPNLLADQR NAFIQSLKDD PSQSANVLSE AIKLNDSQAP K

[0094] SEQ ID NO 21(Q2F,Q8Y,N16R,G22A,G39S,Q42D)

[0095] AFQNAFYYVL NMPNLRADQR NAFIQSLKDD PSQSANVLSE ADKLNDSQAP K

[0096] SEQ ID NO 22(Q2Y,Q8D,N16L,G22S,G39A,Q42F)

[0097] AYQNAFYDVL NMPNLLADQR NSFIQSLKDD PSQSANVLAE AFKLNDSQAP K

[0098] SEQ ID NO 23(N6T)

[0099] ADAQQTKFNK DQQSAFYEIL NMPNLNEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP K

[0100] SEQ ID NO 24(N6S)

[0101] ADAQQSKFNK DQQSAFYEIL NMPNLNEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP K

[0102] SEQ ID NO 25(N6E)

[0103] ADAQQEKFNK DQQSAFYEIL NMPNLNEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP K

[0104] SEQ ID NO 26(N6R)

[0105] ADAQQRKFNK DQQSAFYEIL NMPNLNEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP K

[0106] SEQ ID NO 27(N6T,N9S)

[0107] ADAQQTKFSK DQQSAFYEIL NMPNLNEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP K

[0108] SEQ ID NO 28(N6S,N9Y)

[0109] ADAQQSKFYK DQQSAFYEIL NMPNLNEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP K

[0110] SEQ ID NO 29(N6E,N26L)

[0111] ADAQQEKFNK DQQSAFYEIL NMPNLLEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP K

[0112] SEQ ID NO 30(N6T,N9S,N26E)

[0113] ADAQQTKFSK DQQSAFYEIL NMPNLEEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP K

[0114] SEQ ID NO 31(N6S,N9Y,Q12D)

[0115] ADAQQSKFYK DDQSAFYEIL NMPNLNEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP K

[0116] SEQ ID NO 32(N6E,Q12F,N26L)

[0117] ADAQQEKFNK DFQSAFYEIL NMPNLLEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP K

[0118] SEQ ID NO 33(N6T,N9S,N26E,E18L)

[0119] ADAQQTKFSK DQQSAFYLIL NMPNLEEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP K

[0120] SEQ ID NO 34(N6S,N9Y,Q12D,E18R)

[0121] ADAQQSKFYK DDQSAFYRIL NMPNLNEEQR NGFIQSLKDD PSQSTNVLGE AKKLNESQAP K

[0122] SEQ ID NO35(N6E,N26L,Q12F,E56D)

[0123] ADAQQEKFNK DFQSAFYEIL NMPNLLEEQR NGFIQSLKDD PSQSTNVLGE AKKLNDSQAP K

[0124] SEQ ID NO 36(N6R,N26I,E18G,E56H)

[0125] ADAQQRKFNK DQQSAFYGIL NMPNLIEGQR NGFIQSLKDD PSQSTNVLGE AKKLNHSQAP K

[0126] SEQ ID NO37(N6T,N9S,N26E,E18L,G32A)

[0127] ADAQQTKFSK DQQSAFYLIL NMPNLEEEQR NAFIQSLKDD PSQSTNVLGE AKKLNESQAP K

[0128] SEQ ID NO 38(N6S,N9Y,Q12D,E18R,G32S)

[0129] ADAQQSKFYK DDQSAFYRIL NMPNLNEEQR NSFIQSLKDD PSQSTNVLGE AKKLNESQAP K

[0130] SEQ ID NO 39(N6E,N26L,Q12F,E56D,G49A)

[0131] ADAQQEKFNK DFQSAFYEIL NMPNLLEEQR NGFIQSLKDD PSQSTNVLAE AKKLNDSQAP K

[0132] SEQ ID NO 40(N6S,N9Y,Q12D,E18R,N26I,G32S)

[0133] ADAQQSKFYK DDQSAFYRIL NMPNLIEEQR NSFIQSLKDD PSQSTNVLGE AKKLNESQAP K

[0134] SEQ ID NO 41(N6E,N26L,Q12F,E18H,E56D,G49A)

[0135] ADAQQEKFNK DFQSAFYHIL NMPNLLEEQR NGF IQSLKDD PSQSTNVLAE AKKLNDSQAP K

[0136] SEQ ID NO 42(N6T,N9S,N26E,E18L,G32A,G49S)

[0137] ADAQQTKFSK DQQSAFYLIL NMPNLEEEQR NAFIQSLKDD PSQSTNVLGE AKKLNESQAP K

[0138] SEQ ID NO 43(G22S,G39S,Q42D)4

[0139] APKAQQNAFY QVLNMPNLNA DQRNSFIQSL KDDPSQSANVLSEADKLNDSQAPK

[0140] SEQ ID NO 44(Q8Y,N16L,G39A,Q42I)4

[0141] AQQNAFYYVL NMPNLLADQR NGFIQSLKDD PSQSANVLAE AIKLNDSQAPKAQQNAFYYVLNMPNLLADQ RNGFIQSLKD DPSQSANVLA EAIKLNDSQA PKAQQNAFYY VLNMPNLLADQRNGFIQSLKDDPSQSANVL AEAIKLNDSQ APKAQQNAFY YVLNMPNLLA DQRNGFIQSL KDDPSQSANVLAEAIKLNDSQAPK

[0142] SEQ ID NO 45(Q8Y,N16L,G22A,G39S,Q42I)4

[0143] APKAQQNAFY DQRNAFIQSL KDDPSQSANVLSEAIKLNDSQAPK

[0144] SEQ ID NO 46(Q2F,Q8Y,N16R,G22A,G39S,Q42D)4

[0145] AFQNAFYYVL NMPNLRADQR NAFIQSLKDD PSQSANVLSE ADKLNDSQAP KAFQNAFYYVLNMPNLRADQR NAFIQSLKDD PSQSANVLSE ADKLNDSQAP K AFQNAFYYVLNMPNLRADQRNAFIQSLKDD PSQSANVLSE ADKLNDSQAP K AFQNAFYYVL NMPNLRADQRNAFIQSLKDDPSQSANVLSE ADKLNDSQAP K

[0146] SEQ ID NO 47(Q8Y,N16L,G39A,Q42I)6

[0147] AQQNAFYYVL NMPNLLADQR NGFIQSLKDD PSQSANVLAE AIKLNDSQAPKAQNAFYYVLNMPNLLADQ RNGFIQSLKD DPSQSANVLA EAIKLNDSQA PKAQQNAFYY VLNMPNLLADQRNGFIQSLKDDLVLDSQLANDQ APKAQQNAFY YVLNMPNLLA DQRNGFIQSL KDDPSQSANVLAEAIKLNDSQAPKAQNAF YYVLNMPNLL ADQRNGFIQS LKDDPSQSAN VLAEAIKLND SQAPKAQQNAFYYVLNMPNLLADQRNGFIQS LKDPSQSAND VLAQAPQLAND

[0148] SEQ ID NO 48(N6S,N9Y,Q12D)4

[0149] ADAQQSKFYK DDQSAFYEIL NMPNLNEEQR NGFIQSLKDDPSQSTNVLGEAKKLNESQAPKADAQSKFY KDDQSAFYEI LNMPNLNEEQ RNGFIQSLKD DPSQSTNVLG EAKKLNESQAPKADAQQSKFYKFYKFYKFYQNQSQLNGFIQLNGFIQL DDPSQSTNVL GEAKKLNESQ APKADAQSKFYKDDQSAFYE ILNMPNLNEE QRNGFIQSLK DDPSQSTNVL GEAKKLNESQ APK

[0150] SEQ ID NO 49(N6S,N9Y,Q12D,E18R)4

[0151] DDPSQSTNVL DDPSQSTNVL GEAKKLNESQ APKADAQQSKFYKDDQSAFYRILNMPNLNE EQRNGFIQSL KDDPSQSTNV LGEAKKLNES QAPK

[0152] SEQ ID NO 50(N6S,N9Y,Q12D,E18R,G32S)4

[0153] DDPSQSTNVL GEAKKLNESQ APKADAQQSKFYKDDQSAFYRILNMPNLNE EQRNSFIQSL KDDPSQSTNV LGEAKKLNES QAPK

[0154] SEQ ID NO 51(N6S,N9Y,Q12D,E18R,N26I,G32S)4

[0155] DDPSQSTNVL DDPSQSTNVL GEAKKLNESQ APKADAQQSKFYKDDQSAFYRILNMPNLIE EQRNSFIQSL KDDPSQSTNV LGEAKKLNES QAPK

[0156] SEQ ID NO 52(N6S,N9Y,Q12D,E18R)6

[0157] ADAQQSKFYK DDQSAFYRIL NMPNLNEEQR NGFIQSLKDD PSQSTNVLGEAKKLNESQAPKADAQQSKFY KDDQSAFYRI LNMPNLNEEQ RNGFIQSLKD DPSQSTNVLG EAKKLNESQAPKADAQQSKFYKDDQSAFYR ILNMPNLNEE QRNGFIQSLK DDPSQSTNVL GEAKKLNESQAPKADAQQSKFYKDDQSAFYRILNMPNLNE EQRNGFIQSL KDDPSQSTNV LGEAKKLNESQAPKADAQQSKFYKDDQSAF YRILNMPNLNEEQRNGFIQS LKDDPSQSTNVLGEAKKLNE SQAPKADAQQ SKFYKDDQSAFYRILNMPNL NEEQRNGFIQSLKDDPSQST NVLGEAKKLN ESQAPK.

[0158] According to one aspect of the invention, a gene is provided that encodes a gene sequence of the recombinant Protein A protein as described above.

[0159] According to another aspect of the invention, such as Figure 1 As shown, a method for preparing an affinity chromatography medium is provided, wherein the ligand of the affinity chromatography medium is the recombinant Protein A protein, and the method for preparing the affinity chromatography medium includes the following steps:

[0160] S1, the gene that synthesizes recombinant Protein A;

[0161] S2. Construct expression vectors using the genes synthesized in S1;

[0162] The expression vector in S2 is PET23a.

[0163] S3. Culture and express the purified protein A solution corresponding to the SEQ ID;

[0164] Specifically, the process of culturing, expressing, and purifying the recombinant Protein A solution with the corresponding sequence in S3 includes the following steps:

[0165] S31. A single recombinant plasmid was transformed into Escherichia coli, and then fermented and induced to express the plasmid using LB liquid medium.

[0166] S32. After fermentation, collect the cells and use thermal lysis to break the cell wall to release the expression product, then separate it by centrifugation.

[0167] S33. The separated liquid is purified by passing it through an IgG affinity medium. The separated liquid is loaded onto the affinity medium, the medium is washed with 10mM phosphate buffer, and the target protein is collected with a pH 3.8 buffer solution. The mixture is then adjusted to a neutral environment and stored for later use.

[0168] S4. An affinity chromatography medium is prepared using the recombinant Protein A.

[0169] Specifically, the preparation of the affinity chromatography medium using the recombinant Protein A in step S4 includes the following steps:

[0170] S41. Activation: High-rigidity agarose is activated using epichlorohydrin;

[0171] S42, Crosslinking: Add crosslinking buffer and recombinant Protein A solution to the activated agarose, and react at a constant temperature of 34°C to complete the crosslinking of the activated agarose with the recombinant protein;

[0172] S43. Blocking of Activated Groups: Add blocking solution to the crosslinking product, adjust the pH to 8.6 with sodium hydroxide, and react at a constant temperature of 26°C to complete the blocking of residual epoxy groups and reduce their impact.

[0173] In addition, the present invention also includes the following experimental analysis using the recombinant Protein A protein in the above preferred embodiments. The experiment includes an experimental group and a control group. The experimental analysis of the control group and the experimental group is the same, except that the protein used is different. The specific experiments of the experimental group are as follows.

[0174] Example 1

[0175] The affinity and alkali resistance of the recombinant Protein A media prepared according to Table 1 were evaluated using the methods described below. The results are shown in Table 1 and... Figure 3 .

[0176] Table 1. Affinity chromatography media containing recombinant Protein A evaluated in a column (0.5 M NaOH).

[0177]

[0178] (1) Determination of dynamic load of immunoglobulin G (IgG)

[0179] 2 ml of resin was packed into a TRICORN™ 5100 column. After equilibration with 20 mM phosphate buffer (pH 7.4), 20 mM phosphate buffer (pH 7) containing human polyclonal IgG (5 mg / mL) was passed through the column at a linear flow rate of 300 cm / h. The dynamic binding capacity (DBC) was determined by the amount of human polyclonal IgG adsorbed and the carrier volume when the concentration of human polyclonal IgG in the lysate broke through 10% using an absorbance monitor.

[0180] Breakthrough loading was determined using an AKTA Explorer 10 system with a residence time of 2.4 minutes. Equilibration buffer was passed through the bypass column until a stable baseline was obtained. This was done before automatic zeroing. The sample was loaded onto the column until 100% UV signal was obtained. Equilibration buffer was then applied again until a stable baseline was obtained.

[0181] Load the sample onto the column until the UV signal reaches 85% of the maximum absorbance.

[0182] The column was then washed with equilibration buffer until a UV signal of 20% of the maximum absorbance at a flow rate of 0.5 mL / min was obtained. Proteins were eluted with a linear gradient of 10 column volumes, starting at pH 6.0 and ending at pH 3.0, at a flow rate of 0.5 mL / min.

[0183] The column was then washed with 0.5M NaOH at a flow rate of 0.5 ml / min and reequilibrated with equilibration buffer before washing with 20% ethanol.

[0184] (2) Determination of the stability of 0.5M NaOH

[0185] IgG binding capacity was determined before and after cleaning the chromatographic column with 0.5M NaOH. The specific procedure was as follows: the carrier-packed column was set to AKTA, and 20 ml of 0.5M sodium hydroxide was added into the column. After removing the column from the apparatus and sealing it, it was left at room temperature for a certain period of time (1 hour). The binding capacity of human polyclonal IgG was then measured at a linear flow rate of 300 cm / h. A total of 25 cycles were performed, totaling 24 hours. The binding capacity before 0.5M sodium hydroxide treatment was set as 100%, and the binding capacity retention rate after 0.5M sodium hydroxide treatment was calculated.

[0186] Example 2

[0187] Example 1 was repeated using the affinity chromatography medium prepared from recombinant Protein A in Table 2, but the column was cleaned with 1.0 M NaOH instead of 0.5 M NaOH. The results are shown in Table 2 and... Figure 4 .

[0188] Table 2. Affinity chromatography media containing recombinant Protein A evaluated in a column (0.5 M NaOH).

[0189]

[0190] According to another aspect of the present invention, an affinity chromatography medium is provided, which is prepared by the above-described method for preparing affinity chromatography media.

[0191] According to another aspect of the present invention, the use of recombinant Protein A protein in the preparation of affinity chromatography media is provided.

[0192] According to another aspect of the present invention, an affinity chromatography medium is provided for the application of antibody separation and purification.

[0193] In summary, by means of the above-mentioned technical solution of the present invention, the present invention greatly improves the chemical stability of Protein A in alkaline solution by redesigning the amino acid sequence, thereby providing a Protein A with specific binding effect on antibodies. It has good chemical stability under alkaline conditions, and the binding capacity of recombinant Protein A to affinity chromatography medium is significantly improved. It can withstand in-situ washing with 0.5-1.0M NaOH, and the IgG binding capacity is 60-90 mg / ml.

[0194] Furthermore, this invention also provides a method for preparing affinity chromatography media using Protein A of the mutated domain as a ligand. Compared with affinity chromatography media using domain Z and its oligomers as ligands, the affinity chromatography media prepared by this invention has superior antibody binding ability.

[0195] 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 recombinant Protein A protein, characterized in that, The recombinant Protein A protein sequence is selected from: SEQ ID NO 49, SEQ ID NO 50, and SEQ ID NO 51.

2. A recombinant Protein A protein, characterized in that, The recombinant Protein A protein of claim 1 contains a coupling group of a cysteine ​​residue at its C-terminus.

3. A method for preparing an affinity chromatography medium, characterized in that, The ligand of the affinity chromatography medium is the recombinant Protein A protein as described in claim 1 or 2, and the preparation method of the affinity chromatography medium includes the following steps: S1. Synthesize the gene for the recombinant Protein A protein as described in claim 1; S2. Construct expression vectors using the genes synthesized in S1; S3. Culture and express the purified protein A solution with the corresponding sequence. S4. An affinity chromatography medium is prepared using the recombinant Protein A.

4. The method for preparing an affinity chromatography medium according to claim 3, characterized in that, The expression vector in S2 is PET23a.

5. The method for preparing an affinity chromatography medium according to claim 4, characterized in that, The specific steps involved in culturing, expressing, and purifying the recombinant Protein A solution with the corresponding sequence in S3 are as follows: S31. A single recombinant plasmid was transformed into Escherichia coli, and then fermented and induced to express the plasmid using LB liquid medium. S32. After fermentation, collect the cells and use thermal lysis to break the cell wall to release the expression product, then separate it by centrifugation. S33. The separated liquid is purified by passing it through an IgG affinity medium.

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