A recombinant protein L protein and a preparation method and application thereof

Abstract

The application provides a recombinant Protein L protein and a preparation method and application thereof. The recombinant Protein L protein is a mutant of a Protein L protein domain B5, and is characterized in that the mutant is 1-6 repeat units, each repeat unit is obtained by mutation of an amino acid sequence of the B5 domain, and the mutation position is one or multiple positions of 18, 53 and 68 of the B5 domain. The application selects a strong alkali-resistant domain, the B5 domain of the Protein L protein as a template, and through mutation of amino acids, the protein has strong alkali resistance in a protein purification process, and the purification efficiency is improved.

Description

Technical Field

[0001] This invention belongs to the field of affinity chromatography in biotechnology, specifically relating to a Protein L-mutated immunoglobulin-binding domain with improved chemical stability under alkaline conditions, which can be used in affinity chromatography of immunoglobulins. Background Technology

[0002] Over the past 20 years, compared to traditional radiotherapy and chemotherapy, monoclonal antibodies (MABs) have become one of the most successful cancer treatments. Antibody drugs have consistently accounted for approximately 50% of total biopharmaceutical sales in the last decade. However, antibody purification faces numerous challenges, making it a focus of ongoing research. As research has deepened, new small-molecule antibodies have been discovered to play crucial roles in disease treatment; these new antibodies are called antibody fragments. Because antibody fragments have smaller molecular weights than intact antibodies, they possess better tissue permeability, enabling them to penetrate tissues inaccessible to intact antibodies and exert therapeutic effects. Therefore, antibody fragments have become a hot research topic, and the demand for them is increasing. However, antibody fragments lack an Fc region and cannot be purified using Protein A and Protein G affinity chromatography like intact antibodies. Therefore, it is necessary to find affinity ligands that can effectively bind to antibody fragments.

[0003] Protein L originates from the cell surface of *Peptostreptococcus*, which can be divided into two strains: 312 and 3316. Protein L derived from *Peptostreptococcus* strain 312 has five domains that bind to the kappa light chain of antibody proteins. These domains share high amino acid sequence similarity and can individually bind to antibody proteins. Protein L can bind to the kappa light chain of immunoglobulins, enabling the purification of antibody fragments lacking the Fc structure, including various types of antibodies containing the κ light chain such as IgG, IgM, IgA, IgE, and IgD. This provides a selection option for the purification of antibody fragments lacking the Fc structure. Therefore, Protein L has attracted much attention in antibody fragment drug purification. However, the ligands in currently commercially available Protein L affinity chromatography media are natural Protein L, which cannot withstand in-situ washing with alkaline solutions such as NaOH during purification, exhibiting very limited alkali resistance and problems such as ligand leakage. This results in defects such as short lifespan, low binding capacity, and low purification efficiency, failing to meet the high demands of antibody fragment purification. However, researchers have conducted extensive studies on the alkali stability of Protein A, which is also an affinity chromatography medium. For example, patent 2007 / 097361 reports that by substituting specific amino acid residues in the C or Z domains of Protein A, it can acquire excellent Ig binding ability and alkali stability. Additionally, patent 2000 / 023580 reports that by replacing the asparagine residues in Protein A with other amino acids, it can acquire alkali stability. However, existing reports on improving the alkali stability of Protein L are very rare, and this technology has not been fully studied both domestically and internationally. Summary of the Invention

[0004] The purpose of this invention is to provide a recombinant Protein L protein, its preparation method, and its applications. This invention selects a highly alkali-resistant domain, using the B5 domain of the Protein L protein as a template. Based on this, amino acid mutations are performed to enhance the protein's alkali resistance during purification, thereby improving purification efficiency.

[0005] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows:

[0006] The present invention provides a recombinant Protein L protein, which is a mutant of the Protein L protein domain B5. The mutant consists of 1-6 repeating units, each of which is obtained by mutating the amino acid sequence of the B5 domain. The mutation position is one or more of the 18th, 53rd and 68th positions of the B5 domain.

[0007] In a preferred embodiment, the mutation mode of position 18 of the B5 domain is: asparagine is mutated to alanine, glutamine, aspartic acid, or valine; the mutation mode of position 53 of the B5 domain is: histidine is mutated to alanine; and the mutation mode of position 68 of the B5 domain is: asparagine is mutated to alanine, glutamine, aspartic acid, or histidine.

[0008] In a preferred embodiment, the mutation mode of each repeating unit of the mutant is one of the following:

[0009] (1) Mutations at positions 18 and 68 of the B5 domain, wherein the asparagine at position 18 of the B5 domain is mutated to alanine and the asparagine at position 68 of the B5 domain is mutated to glutamine.

[0010] (2) Mutations at positions 18 and 68 of the B5 domain, wherein the asparagine at position 18 of the B5 domain is mutated to glutamine, and the asparagine at position 68 of the B5 domain is mutated to glutamine.

[0011] (3) Mutations at positions 18 and 68 of the B5 domain, wherein the asparagine at position 18 of the B5 domain is mutated to glutamine, and the asparagine at position 68 of the B5 domain is mutated to aspartic acid.

[0012] (4) Mutations at positions 18 and 68 of the B5 domain, wherein the asparagine at position 18 of the B5 domain is mutated to aspartic acid and the asparagine at position 68 of the B5 domain is mutated to glutamine.

[0013] (5) Mutations at positions 18, 53 and 68 of the B5 domain, wherein the asparagine at position 18 of the B5 domain is mutated to glutamine, the histidine at position 53 of the B5 domain is mutated to alanine, and the asparagine at position 68 of the B5 domain is mutated to glutamine.

[0014] (6) Mutations at positions 18, 53 and 68 of the B5 domain, wherein the asparagine at position 18 of the B5 domain is mutated to aspartic acid, the histidine at position 53 of the B5 domain is mutated to alanine, and the asparagine at position 68 of the B5 domain is mutated to glutamine.

[0015] (7) Mutations at positions 18, 53 and 68 of the B5 domain, wherein the asparagine at position 18 of the B5 domain is mutated to valine, the histidine at position 53 of the B5 domain is mutated to alanine, and the asparagine at position 68 of the B5 domain is mutated to glutamine.

[0016] In a preferred embodiment, the mutant consists of four repeating units.

[0017] The present invention also provides a polynucleotide encoding the recombinant Protein L protein.

[0018] The present invention also provides a carrier comprising the aforementioned polynucleotide.

[0019] The present invention also provides a transformant comprising the aforementioned carrier.

[0020] The present invention also provides a chromatography medium comprising a solid support and the recombinant Protein L protein bound thereto.

[0021] The present invention also provides the application of the chromatographic medium for separating antibodies or fragments thereof.

[0022] In a preferred embodiment, the antibody is a protein containing a κ light chain.

[0023] The present invention also provides a method for preparing recombinant Protein L protein, comprising expressing the recombinant Protein L protein in the transformant or a cell-free protein synthesis system, or chemically synthesizing it.

[0024] In some embodiments, the disclosed polypeptides and / or polymers further include one or more coupling elements at the C-terminus or N-terminus, selected from cysteine ​​residues, multiple lysine residues, and multiple histidine residues. The coupling element can, for example, be a single C-terminal cysteine ​​residue. The coupling element can be directly attached to the C-terminus or N-terminus. Having a C-terminal or N-terminal cysteine ​​residue is advantageous because terminal coupling of the protein can be accomplished through the reaction of cysteine ​​thiol with an electrophilic group on the support.

[0025] The sequences involved in this invention are as follows:

[0026] SEQ ID NO: 1 (B1 domain)

[0027] KEETPETPET DSEEEVTIKA NLIFANGSTQ TAEFKGTFEK ATSEAYAYAD TLKKDNGEYTVDVADKGYTL NIKFAG

[0028] SEQ ID NO: 2 (B2 domain)

[0029] KEKTPEEPKE EVTIKANLIY ADGKTQTAEF KGTFEEATAE AYRYADALKK DNGEYTVDVADKGYTLNIKF AG

[0030] SEQ ID NO: 3 (B3 domain)

[0031] KEKTPEEPKE EVTIKANLIY ADGKTQTAEF KGTFEEATAE AYRYADLLAK ENGKYTVDVADKGYTLNIKF AG

[0032] SEQ ID NO:4(B4 domain)

[0033] KEKTPEEPKE EVTIKANLIY ADGKTQTAEF KGTFAEATAE AYRYADLLAK ENGKYTADLEDGGYTINIRF AG

[0034] SEQ ID NO:5(B5 domain)

[0035] KKVDEKPEEK EQVTIKENIY FEDGTVQTAT FKGTFAEATA EAYRYADLLS KEHGKYTADLEDGGYTINIR FAG

[0036] SEQ ID NO:6(N18A)

[0037] KKVDEKPEEK EQVTIKEAIY FEDGTVQTAT FKGTFAEATA EAYRYADLLS KEHGKYTADLEDGGYTINIR FAG

[0038] SEQ ID NO:7(N18Q)

[0039] KKVDEKPEEK EQVTIKEQIY FEDGTVQTAT FKGTFAEATA EAYRYADLLS KEHGKYTADLEDGGYTINIR FAG

[0040] SEQ ID NO:8(N18D)

[0041] KKVDEKPEEK EQVTIKEDIY FEDGTVQTAT FKGTFAEATA EAYRYADLLS KEHGKYTADLEDGGYTINIR FAG

[0042] SEQ ID NO:9(N18V)

[0043] KKVDEKPEEK EQVTIKEVIY FEDGTVQTAT FKGTFAEATA EAYRYADLLS KEHGKYTADLEDGGYTINIR FAG

[0044] SEQ ID NO:10(H53A)

[0045] KKVDEKPEEK EQVTIKENIY FEDGTVQTAT FKGTFAEATA EAYRYADLLS KEAGKYTADLEDGGYTINIR FAG

[0046] SEQ ID NO:11(N68A)

[0047] KKVDEKPEEK EQVTIKENIY FEDGTVQTAT FKGTFAEATA EAYRYADLLS KEHGKYTADLEDGGYTIAIR FAG

[0048] SEQ ID NO:12(N68Q)

[0049] KKVDEKPEEK EQVTIKENIY FEDGTVQTAT FKGTFAEATA EAYRYADLLS KEHGKYTADLEDGGYTIQIR FAG

[0050] SEQ ID NO:13(N68D)

[0051] KKVDEKPEEK EQVTIKENIY FEDGTVQTAT FKGTFAEATA EAYRYADLLS KEHGKYTADLEDGGYTIDIR FAG

[0052] SEQ ID NO:14(N18A,N68Q)

[0053] KKVDEKPEEK EQVTIKEAIY FEDGTVQTAT FKGTFAEATA EAYRYADLLS KEHGKYTADLEDGGYTIQIR FAG

[0054] SEQ ID NO:15(N18Q,N68A)

[0055] KKVDEKPEEK EQVTIKEQIY FEDGTVQTAT FKGTFAEATA EAYRYADLLS KEHGKYTADLEDGGYTIAIR FAG

[0056] SEQ ID NO:16(N18Q,N68Q)

[0057] KKVDEKPEEK EQVTIKEQIY FEDGTVQTAT FKGTFAEATA EAYRYADLLS KEHGKYTADLEDGGYTIQIR FAG

[0058] SEQ ID NO: 17(N18Q, N68D)

[0059] KKVDEKPEEK EQVTIKEQIY FEDGTVQTAT FKGTFAEATA EAYRYADLLS KEHGKYTADLEDGGYTIDIR FAG

[0060] SEQ ID NO: 18(N18D, N68Q)

[0061] KKVDEKPEEK EQVTIKEDIY FEDGTVQTAT FKGTFAEATA EAYRYADLLS KEHGKYTADLEDGGYTIQIR FAG

[0062] SEQ ID NO:19(N18Q,H53A,N68Q)

[0063] KKVDEKPEEK EQVTIKEQIY FEDGTVQTAT FKGTFAEATA EAYRYADLLS KEAGKYTADLEDGGYTIQIR FAG

[0064] SEQ ID NO:20(N18D,H53A,N68Q)

[0065] KKVDEKPEEK EQVTIKEDIY FEDGTVQTAT FKGTFAEATA EAYRYADLLS KEAGKYTADLEDGGYTIQIR FAG

[0066] SEQ ID NO:21(N18V,H53A,N68Q)

[0067] KKVDEKPEEK EQVTIKEVIY FEDGTVQTAT FKGTFAEATA EAYRYADLLS KEAGKYTADLEDGGYTIQIR FAG

[0068] SEQ ID NO: 22(B1 domain)4

[0069] KEETPETPET DSEEEVTIKA NLIFANGSTQ TAEFKGTFEK ATSEAYAD TLKKDNGEYTVDVADKGYTL NIKFAGKEET PETPETDSEE EVTIKANLIF ANGSTQTAEF KGTFEKATSE AYADTLKDNGEYTDTLEEKGTFEK DKK KANLIFANGS TQTAEFKGTF EKATSEAYADTLKKDNGE YTVDVADKGY TLNIKFAGKE ETPETPETDS EEEVTIKANL IFANGSTQTA EFKGTFEKATSEAYADTL KKDNGEYTVD VADKGYTLNI KFAG

[0070] SEQ ID NO:23(B2 domain)4

[0071] KEKTPEEPKE EVTIKANLIY ADGKTQTAEF KGTFEEATAE AYRYADKK DNGEYTVDVADKGYTLNIKF AGKEKTPEEP KEEVTIKANL IYADGKTQTA EFKGTFEEAT AEYRYADAL KKDNGFAKYTLTVDE EPKEEVTIKA NLIYADGKTQ TAEFKGTFEE ATAEAYRYAD ALKKDNGEYT VDVADKGYTL NIKFAGKEKT PEEPKEEVTI KANLIYADGK TQTAEFKGTF EEATAEAYRYADKKDNGY YTVDVADKGGY

[0072] SEQ ID NO:24(B3 domain)4

[0073] KEKTPEEPKE EVTIKANLIY ADGKTQTAEF KGTFEEATAE AYRYADLLAK ENGKYTVDVADKGYTLNIKF AGKEKTPEEP KEEVTIKANL IYADGKTQTA EFKGTFEEAT AEAYRYADLL AKENGKYTVDVADKGYTLNI KFAGKEKTPE EPKEEVTIKA NLIYADGKTQ TAEFKGTFEE ATAEAYRYAD LLAKENGKYTVDVADKGYTL NIKFAGKEKT PEEPKEEVTI KANLIYADGK TQTAEFKGTF EEATAEAYRY ADLLAKENGKYTVDVADKGY TLNIKFAG

[0074] SEQ ID NO:25(B4 domain)4

[0075] KEKTPEEP EVTIKANLIY ADGKTQTAEF KGTFAEATAE AYRYADLLAK ENGKYTADLEDGGYTINIRF AGKEKTPEEP KEEVTIKANL IYADGKTQTA EFKGTFAEAT AEAYRYADLL AKENGKYTADLEDGGYTINI RFAGKEKTPE EPKEEVTIKA NLIYADGKTQ TAEFKGTFAE ATAEAYRYAD LLAKENGKYTADLEDGGYTI NIRFAGKEKT PEEPKEEVTI KANLIYADGK TQTAEFKGTF AEATAEAYRY ADLLAKENGKYTADLEDGGY TINIRFAG

[0076] SEQ ID NO:26(B5 domain)4

[0077] KKVDEKPEEK EQVTIKENIY FEDGTVQTAT FKGTFAEATA EAYRYADLLS KEHGKYTADLEDGGYTINIR FAGKKVDEKP EEKEQVTIKE NIYFEDGTVQ TATFKGTFAE ATAEAYRYAD LLSKEHGKYTADLEDGGYTI NIRFAGKKVD EKPEEKEQVT IKENIYFEDG TVQTATFKGT FAEATAEAYR YADLLSKEHGKYTADLEDGG YTINIRFAGK KVDEKPEEKE QVTIKENIYF EDGTVQTATF KGTFAEATAE AYRYADLLSKEHGKYTADLE DGGYTINIRF AG

[0078] SEQ ID NO: 27(N18A)4

[0079] KKVDEKPEEK EQVTIKEAIY FEDGTVQTAT FKGTFAEATA EAYRYADLLS KEHGKYTADLEDGGYTINIR FAGKKVDEKP EEKEQVTIKE AIYFEDGTVQ TATFKGTFAE ATAEAYRYAD LLSKEHGKYTADLEDGGYTI NIRFAGKKVD EKPEEKEQVT IKEAIYFEDG TVQTATFKGT FAEATAEAYR YADLLSKEHGKYTADLEDGG YTINIRFAGK KVDEKPEEKE QVTIKEAIYF EDGTVQTATF KGTFAEATAE AYRYADLLSKEHGKYTADLE DGGYTINIRF AG

[0080] SEQ ID NO: 28(N18Q)4

[0081] KKVDEKPEEK EQVTIKEQIY FEDGTVQTAT FKGTFAEATA EAYRYADLLS KEHGKYTADLEDGGYTINIR FAGKKVDEKP EEKEQVTIKE QIYFEDGTVQ TATFKGTFAE ATAEAYRYAD LLSKEHGKYTADLEDGGYTI NIRFAGKKVD EKPEEKEQVT IKEQIYFEDG TVQTATFKGT FAEATAEAYR YADLLSKEHGKYTADLEDGG YTINIRFAGK KVDEKPEEKE QVTIKEQIYF EDGTVQTATF KGTFAEATAE AYRYADLLSKEHGKYTADLE DGGYTINIRF AG

[0082] SEQ ID NO: 29(N18D)4

[0083] KKVDEKPEEK EQVTIKEDIY FEDGTVQTAT FKGTFAEATA EAYRYADLLS KEHGKYTADLEDGGYTINIR FAGKKVDEKP EEKEQVTIKE DIYFEDGTVQ TATFKGTFAE ATAEAYRYAD LLSKEHGKYTADLEDGGYTI NIRFAGKKVD EKPEEKEQVT IKEDIYFEDG TVQTATFKGT FAEATAEAYR YADLLSKEHGKYTADLEDGG YTINIRFAGK KVDEKPEEKE QVTIKEDIYF EDGTVQTATF KGTFAEATAE AYRYADLLSKEHGKYTADLE DGGYTINIRF AG

[0084] SEQ ID NO: 30(N18V)4

[0085] KKVDEKPEEK EQVTIKEVIY FEDGTVQTAT FKGTFAEATA EAYRYADLLS KEHGKYTADLEDGGYTINIR FAGKKVDEKP EEKEQVTIKE VIYFEDGTVQ TATFKGTFAE ATAEAYRYAD LLSKEHGKYTADLEDGGYKKPEKTIKEVIKQDQDVKEVKE IKEVIYFEDG TVQTATFKGT FAEATAEAYR YADLLSKEHGKYTADLEDGG YTINIRFAGK KVDEKPEEKE QVTIKEV IYF EDGTVQTATF KGTFAEATAE AYRYADLLSKEHGKYTADLE DGGYTINIRF AG

[0086] SEQ ID NO:31(H53A)4

[0087] KKVDEKPEEK EQVTIKENIY FEDGTVQTAT FKGTFAEATA EAYRYADLLS KEAGKYTADLEDGGYTINIR FAGKKVDEKP EEKEQVTIKE NIYFEDGTVQ TATFKGTFAE ATAEAYRYAD LLSKEAGKYTADLEDGGYTI NGPEKVEKVQDGGYTINIR IKENIYFEDG TVQTATFKGT FAEATAEAYR YADLLSKEAGKYTADLEDGG YTINIRFAGK KVDEKPEEKE QVTIKENIYF EDGTVQTATF KGTFAEATAEA YRYADLLSKEAGKYTADLE DGGYTINIRF AG

[0088] SEQ ID NO:32(N68A)4

[0089] KKVDEKPEEK EQVTIKENIY FEDGTVQTAT FKGTFAEATA EAYRYADLLS KEHGKYTADLEDGGYTIAIR FAGKKVDEKP EEKEQVTIKE NIYFEDGTVQ TATFKGTFAE ATAEAYRYAD LLSKEHGKYTADLEDGGYTI AIRFAGKKVD EKPEEKEQVT IKENIYFEDG TVQTATFKGT FAEATAEAYR YADLLSKEHGKYTADLEDGG YTIAIRFAGK KVDEKPEEKE QVTIKENIYF EDGTVQTATF KGTFAEATAE AYRYADLLSKEHGKYTADLE DGGY TIAIRF A G

[0090] SEQ ID NO:33(N68Q)4

[0091] KKVDEKPEEK EQVTIKENIY FEDGTVQTAT FKGTFAEATA EAYRYADLLS KEHGKYTADLEDGGYTIQIR FAGKKVDEKP EEKEQVTIKE NIYFEDGTVQ TATFKGTFAE ATAEAYRYAD LLSKEHGKYTADLEDGGYTI QIRFAGKKVD EKPEEKEQVT IKENIYFEDG TVQTATFKGT FAEATAEAYR YADLLSKEHGKYTADLEDGG YTIQIRFAGK KVDEKPEEKE QVTIKENIYF EDGTVQTATF KGTFAEATAE AYRYADLLSKEHGKYTADLE DGGYTI QIRF AG

[0092] SEQ ID NO:34(N68D)4

[0093] KKVDEKPEEK EQVTIKENIY FEDGTVQTAT FKGTFAEATA EAYRYADLLS KEHGKYTADLEDGGYTIDIR FAGKKVDEKP EEKEQVTIKE NIYFEDGTVQ TATFKGTFAE ATAEAYRYAD LLSKEHGKYTADLEDGGYTI DIRFAGKKVD EKPEEKEQVT IKENIYFEDG TVQTATFKGT FAEATAEAYR YADLLSKEHGKYTADLEDGG YTIDIRFAGK KVDEKPEEKE QVTIKENIYF EDGTVQTATF KGTFAEATAE AYRYADLLSKEHGKYTADLE DGGYTIDIRF AG

[0094] SEQ ID NO: 35(N18A,N68Q)4

[0095] KKVDEKPEEK EQVTIKEAIY FEDGTVQTAT FKGTFAEATA EAYRYADLLS KEHGKYTADLEDGGYTIQIR FAGKKVDEKP EEKEQVTIKE AIYFEDGTVQ TATFKGTFAE ATAEAYRYAD LLSKEHGKYTADLEDGGYTI QIRFAGKKVD EKPEEKEQVT IKEAIYFEDG TVQTATFKGT FAEATAEAYR YADLLSKEHGKYTADLEDGG YTIQIRFAGK KVDEKPEEKE QVTIKEAIYF EDGTVQTATF KGTFAEATAEA YRYADLLSKEHGKYTADLE DGGYT IQIRF AG

[0096] SEQ ID NO: 36(N18Q,N68A)4

[0097] KKVDEKPEEK EQVTIKEQIY FEDGTVQTAT FKGTFAEATA EAYRYADLLS KEHGKYTADLEDGGYTIAIR FAGKKVDEKP EEKEQVTIKE QIYFEDGTVQ TATFKGTFAE ATAEAYRYAD LLSKEHGKYTADLEDGGYTI AIRFAGKKVD EKPEEKEQVT IKEQIYFEDG TVQTATFKGT FAEATAEAYR YADLLSKEHGKYTADLEDGG YTIAIRFAGK KVDEKPEEKE QVTIKEQIYF EDGTVQTATF KGTFAEATAE AYRYADLLSKEHGKYTADLE DGGYTIAIRF AG

[0098] SEQ ID NO: 37(N18Q,N68Q)4

[0099] KKVDEKPEEK EQVTIKEQIY FEDGTVQTAT FKGTFAEATA EAYRYADLLS KEHGKYTADLEDGGYTIQIR FAGKKVDEKP EEKEQVTIKE QIYFE DGTVQ TATFKGTFAE ATAEAYRYAD LLSKEHGKYTADLEDGGYTI QIRFAGKKVD EKPEEKEQVT IKEQIYFEDG TVQTATFKGT FAEATAEAYR YADLLSKEHGKYTADLEDGG YTIQIRFAGK KVDEKPEEKE QVTIKEQIYF EDGTVQTATF KGTFAEATAE AYRYADLLSKEHGKYTADLE DGGYTIQIRF AG

[0100] SEQ ID NO: 38(N18Q,N68D)4

[0101] KKVDEKPEEK EQVTIKEQIY FEDGTVQTAT FKGTFAEATA EAYRYADLLS KEHGKYTADLEDGGYTIDIR FAGKKVDEKP EEKEQVTIKE QIYFE DGTVQ TATFKGTFAE ATAEAYRYAD LLSKEHGKYTADLEDGGYTI DIR FAGKKVD EKPEEKEQVT IKEQIYFEDG TVQTATFKGT FAEATAEAYR YADLLSKEHG KYTADLEDGG YTIDIRFAGK KVDEKPEEKE QVTIKEQIYF EDGTVQTATF KGTFAEATAEAYRYADLLSK EHGKYTADLE DGGY TIDIRF AG

[0102] SEQ ID NO: 39(N18D,N68Q)4

[0103] KKVDEKPEEK EQVTIKEDIY FEDGTVQTAT FKGTFAEATA EAYRYADLLS KEHGKYTADLEDGGYTIQIR FAGKKVDEKP EEKEQVTIKE DIYFEDGTVQ TATFKGTFAE ATAEAYRYAD LLSKEHGKYTADLEDGGYTI QIRFAGKKVD EKPEEKEQVT IKEDIYFEDG TVQTATFKGT FAEATAEAYR YADLLSKEHGKYTADLEDGG YTIQIRFAGK KVDEKPEEKE QVTIKEDIYF EDGTVQTATF KGTFAEATAE AYRYADLLSKEHGKYTADLE DGGYTIQIRF AG

[0104] SEQ ID NO:40(N18Q,H53A,N68Q)4

[0105] KKVDEKPEEK EQVTIKEQIY FEDGTVQTAT FKGTFAEATA EAYRYADLLS KEAGKYTADLEDGGYTIQIR FAGKKVDEKP EEKEQVTIKE QIYFEDGTVQ TATFKGTFAE ATAEAYRYAD LLSKEAGKYTADLEDGYKKPEEKVQQDVQTVQTVQ IKEQIYFEDG TVQTATFKGT FAEATAEAYR YADLLSKEAGKYTADLEDGG YTIQIRFAGK KVDEKPEEKE QVTIKEQIYF EDGT VQTATF KGTFAEATAE AYRYADLLSKEAGKYTADLE DGG YT IQ IRF AG

[0106] SEQ ID NO:41(N18D,H53A,N68Q)4

[0107] KKVDEKPEEK EQVTIKEDIY FEDGTVQTAT FKGTFAEATA EAYRYADLLS KEAGKYTADLEDGGYTIQIR FAGKKVDEKP EEKEQVTIKE DIYFE DGTVQ TATFKGTFAE ATAEAYRYAD LLSKEAGKYTADLEDGGYTI QVEKVEKVEKVEKVKVEKVKV IKEDIYFEDG TVQTATFKGT FAEATAEAYR YADLLSKEAGKYTADLEDGG YTIQIRFAGK KVDEKPEEKE QVTIKEDIYF EDGTVQTATF KGTFAEATAEAYRYADLLSK EAGKYTADLE DGGYTIQIRF AG

[0108] SEQ ID NO:42(N18V,H53A,N68Q)4

[0109] KKVDEKPEEK EQVTIKEVIY FEDGTVQTAT FKGTFAEATA EAYRYADLLS KEAGKYTADLEDGGYTIQIR FAGKKVDEKP EEKEQVTIKE VIYFEDGTVQ TATFKGTFAE ATAEAYRYAD LLSKEAGKYTADLEDGGYTI QIRFAGKKVD EKPEEKEQVT IKEVIYFEDG TVQTATFKGT FAEATAEAYR YADLLSKEAGKYTADLEDGG YTIQIRFAGK KVDEKPEEKE QVTIKEVIYF EDGTVQTATF KGTFAEATAE AYRYADLLSKEAGKYTADLE DGGYTIQIR F AG

[0110] SEQ ID NO: 43 (N10Q, N45A, N60Q) 4

[0111] PKEEVTIKAN LIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKENGKYTV DVADKGYTLNIKFAGPKEEV TIKANLIYAD GKTQTAEFKGTFEEATAEAY RYADLLAKEN GKYTVDVADK GYTLNIKFAGPKEEV TIKAN LIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKENGKYTV DVADKGYTLN IKFAGPKEEVTIKANLIYAD GKTQTAEFKG TFEEA TAEAY RYADLLAKEN GKYTVDVADK GYTLNIKFAG

[0112] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0113] 1. This invention provides a recombinant Protein L protein, which improves the basic stability of Protein L and its binding ability to antibody proteins by using the B5 domain of Protein L as a template and performing amino acid mutations on it.

[0114] 2. This invention provides a recombinant Protein L protein chromatography medium with high alkali resistance and high loading capacity, allowing it to undergo multiple CIP washing processes without damaging Protein L, thus enabling reuse. This provides an efficient purification method for antibody fragments and intact antibodies, ensuring the safety of antibody drug production in my country. Attached Figure Description

[0115] Figure 1 The images show the SDS-PAGE analysis results of Protein L's B1-B5 domain proteins after alkali treatment in 20mM NaOH for 0h, 8h, and 28h, respectively.

[0116] Figure 2 This is a schematic diagram showing the stability of the (B3 domain)4, (B4 domain)4, and (B5 domain)4 chromatography media of Protein L in this invention against 50 mM NaOH alkali.

[0117] Figure 3 This is a schematic diagram showing the stability of the recombinant Protein L chromatography medium with mutations in the four repeating units of the B5 domain of this invention against 50 mM NaOH alkali. Detailed Implementation

[0118] The technical solution of the present invention will be described in detail below with reference to the embodiments. Unless otherwise specified, all reagents and biological materials used below are commercial products.

[0119] Example 1: Construction, transformation, expression and purification of Protein L mutant

[0120] (1) Construction of Protein L mutant

[0121] The designs, such as SEQ ID NO.1–42 and comparative example SEQ ID NO.43, were constructed into different Protein L plasmid vectors by Shanghai Sangon Biotech Co., Ltd.

[0122] (2) Transformation of Protein L mutant

[0123] The constructed plasmid powder was dissolved in 20 μl of sterile pure water. After thorough dissolution, 2 μl of the dissolved plasmid was added to 100 μl of competent cells. The cell suspension was placed in a flotation plate and incubated on ice for 30 min, followed by heat shock at 42°C for 90 s, and then immediately incubated on ice for 2 min. The cell suspension was spread onto LB agar plates and incubated for 1 h. After incubation, single colonies were picked and inoculated into 5 mL of LB medium (containing kanamycin) and cultured on a shaker at 37°C for 5 h. Single colonies with high induced expression levels were selected, and an equal volume of sterile glycerol was added. The mixture was then aliquoted into sterile cryovials and stored at -70°C for later use.

[0124] (3) Expression and purification of Protein L mutant

[0125] Thaw the frozen bacterial culture, and add 150 μl of the thawed solution to LB medium (containing kanamycin). Incubate at 37°C on a shaker for 5 h. After 5 h, add the bacterial culture back to LB liquid medium (containing kanamycin) and continue incubation at 37°C on a shaker for another 5 h. When the bacterial culture becomes concentrated, add 0.2 mM isopropyl-β-D-thiogalactoside (IPTG) and induce fermentation for 5 h before scaling up the culture. After fermentation, collect the bacterial cells and use thermal lysis to break down the cell walls and release the expression product, which is then separated by centrifugation. Purify the separated liquid using IgG affinity medium. Load the separated liquid onto the affinity medium, wash the medium with 10 mM phosphate buffer, collect the target protein with a pH 3.8 buffer solution, adjust to a neutral environment, and store for later use.

[0126] (4) Preparation of Protein L mutant chromatography media

[0127] The purified Protein L mutant proteins were concentrated using ultrafiltration tubes, with each protein reaching a concentration of at least 10 mg / mL. 10 g of high-rigidity agarose was washed with purified water and dried. 10 g of the agarose was weighed, and 20 mL of purified water, 0.2 g of sodium hydroxide, and 10 mL of epichlorohydrin were added to a 100 mL flask. The mixture was reacted at 27°C for 2 hours. The activated gel was then washed with 1 L of water to obtain activated agarose microspheres. The activated agarose microspheres were added to a 100 mL flask, followed by 150 mg NaHCO3, 10 mg Na2CO3, 150 mg NaCl, and 10 mg EDTA. After stirring evenly, 50 mL of Protein L mutant protein was added, and the mixture was reacted at 34 °C for 8 hours to complete the cross-linking. 100 mL of blocking solution (ethanolamine solution) was added to the cross-linking product, and the pH was adjusted to 8.6 with sodium hydroxide. The mixture was reacted at 26 °C for 2 hours to complete the blocking of the residual epoxy groups.

[0128] (5) Evaluation of the stability of the alkali resistance of Protein L mutant media

[0129] The cross-linked media were packed into columns, with each media packed into 1 mL columns. The loading was first determined using the following method:

[0130] Test method:

[0131] Testing equipment: AKTA Pure

[0132] Sample: IgG (containing only Kappa light chain) 3 mg / mL;

[0133] Chromatography column: 1 mL Protein L affinity chromatography column

[0134] Flow rate: 0.17 mL / min; Detection wavelength: 280 nm;

[0135] Solution A (equilibration solution): PBS; pH: 7.4;

[0136] Solution B (elution buffer): 0.1M HAC; pH: 2.5;

[0137] Before loading the sample, equilibrate the chromatography column with 5CV solution A and zero the UV value. During the loading process, collect the flow-through liquid when the UV peak appears. After loading, elute the chromatography column and collect the eluent in a centrifuge tube when the UV peak appears. Immediately after loading, use a spectrophotometer to measure the OD280 value of the eluent and calculate the corresponding concentration. The elution volume is calculated based on the collected volume given the eluent concentration, thus determining the loading capacity of the medium. Afterward, adjust the pH of all collected solutions to neutral in a timely manner. This determination method follows the method for determining the maximum dynamic loading capacity of 10%.

[0138] The CIP loop works as follows:

[0139] Circulating device: AKTA Explorer

[0140] Chromatography column: Protein L affinity chromatography column, 1 mL (CV)

[0141] Detection wavelength: 280nm;

[0142] Required solution:

[0143] Solution A: PBS; pH: 7.4;

[0144] Solution B: 0.1M HAC; pH: 2.5;

[0145] Solution C: 50mM NaOH

[0146] The load measurement procedure settings are shown in Table 1.

[0147] Table 1

[0148] balance PBS 0.33 3 Cleaning HAC 0.33 5 balance PBS 0.33 3 Alkali treatment NaOH 0.2 3 balance PBS 0.33 3.5

[0149] One cycle consists of equilibration-washing-equilibration-alkali treatment-equilibration. After 25 cycles, the loading is measured and recorded using the loading determination method. Each chromatography column requires 50 alkali-resistant cycles.

[0150] Example 2: Comparative experiment on the alkali resistance of proteins with four repeating units of the B1-B5 domain.

[0151] The four repeating units of the B1-B5 domains are sequence numbers SEQ ID NO.22-SEQ ID NO.26, and the corresponding mutants are named (B1 domain)4, (B2 domain)4, (B3 domain)4, (B4 domain)4, and (B5 domain)4, respectively. Plasmid vectors were constructed by Shanghai Sangon Biotech Co., Ltd., and then the Protein L plasmid vector was transformed. After transformation, fermentation expression was performed to obtain the corresponding expression strains. After purification, the proteins of each domain were subjected to alkali treatment to preliminarily screen for strains with good alkali tolerance. The alkali treatment method is as follows:

[0152] Take 1 mL of protein and add an equal volume of 20 mM sodium hydroxide, mix well, and place in a 23°C low-temperature biochemical incubator. Immediately, take 200 μL of each protein into a 2 mL centrifuge tube, add 100 μL of 0.2 M hydrochloric acid and 40 μL of 1 M PB, vortex to mix, and place in a 4°C refrigerator. This protein is considered the sample at 0 h of alkali treatment. Neutralize the protein solution at 8 h and 28 h of alkali treatment using the same method. Simultaneously, take 100 μL of the alkali-treated protein solution from the three different time periods (0 h, 8 h, 28 h), add 50 μL of reduced 3X Loading Buffer, and mix well. Heat the sample with Loading Buffer for 2 min, and then perform SDS-PAGE analysis to observe the protein decomposition at different time points of alkali treatment. Figure 1 The image shows the SDS-PAGE analysis results of different domains of Protein L (B1-B5) after treatment with 20 mM NaOH for 0 h, 8 h, and 28 h. Figure 1 The results showed that the (B3 domain)4, (B4 domain)4, and (B5 domain)4 proteins were more alkali-resistant than the (B1 domain)4 and (B2 domain)4 proteins; therefore, further alkali resistance studies were conducted on these three proteins.

[0153] Example 3: Preparation and alkali resistance stability evaluation of (B3 domain)4, (B4 domain)4, and (B5 domain)4 chromatography media

[0154] The purified (B3 domain)4, (B4 domain)4, and (B5 domain)4 proteins were concentrated using ultrafiltration to a concentration of at least 10 mg / mL for later use. 10 g of high-rigidity agarose was washed with purified water and dried. 10 g of the agarose was weighed and added to 20 mL of purified water, 0.2 g of sodium hydroxide, and 10 mL of epichlorohydrin in a 100 mL flask. The mixture was reacted at 27°C for 2 hours. The activated gel was washed with 1 L of water to obtain activated agarose microspheres. The activated agarose microspheres were added to a 100 mL flask, followed by 150 mg NaHCO3, 10 mg Na2CO3, 150 mg NaCl, and 10 mg EDTA. After mixing the three groups thoroughly, 50 mL of (B3 domain)4, (B4 domain)4, and (B5 domain)4 proteins were added to each group, and the mixture was reacted at 34 °C for 8 hours to complete the cross-linking. 100 mL of blocking solution (ethanolamine solution) was added to the cross-linking product, the pH was adjusted to 8.6 with sodium hydroxide, and the mixture was reacted at 26 °C for 2 hours to complete the blocking of the residual epoxy groups.

[0155] Then, the alkali resistance of the three protein media (B3 domain)4, (B4 domain)4, and (B5 domain)4 was evaluated using the alkali resistance stability evaluation method for Protein L mutant media described in Example 1 (5). The results of 50 alkali resistance cycles are shown in Table 2 and Figure 2 .

[0156] Table 2

[0157]

[0158] Figure 2 This diagram illustrates the stability of the (B3 domain)4, (B4 domain)4, and (B5 domain)4 chromatographic media of the Protein L mutant protein to 50 mM NaOH. Analysis of the data in Table 2 shows that the (B5 domain)4 chromatographic medium exhibits better alkali resistance than the (B3 domain)4 and (B4 domain)4 protein chromatographic media after 50 cycles of 50 mM NaOH. In other words, the B5 domain of *Streptococcus pyogenes* strain 312 has stronger alkali resistance than other domains. Therefore, the B5 domain was selected for mutation in this invention.

[0159] Example 4: Evaluation of the alkali resistance stability of reconstituted Protein L medium containing B5 domain

[0160] The B5 domain is shown in SEQ ID NO. 5. One or more amino acids at positions 18, 53, and 68 were replaced with other amino acids to synthesize a mutant. The sequence numbers of the prepared mutants are shown in SEQ ID NO. 36-SEQ ID NO. 42. The sequence number of the control group (Cytiva product, B3 domain mutant) is shown in SEQ ID NO. 43. Protein L mutant chromatography media were prepared according to the method described in Example 1 (4). The stability of the Protein L mutant media under alkali resistance was then evaluated using the method described in Example 1 (5). The results of 50 alkali resistance cycles are shown in Table 3 and... Figure 3 .

[0161] Table 3

[0162]

[0163]

[0164] Figure 3 This diagram illustrates the stability of recombinant Protein L chromatography media with mutations in the four repeating units of the B5 domain against 50 mM NaOH. As shown in Table 3, after 50 cycles of alkali resistance, compared to the original unmutated Protein L with the B5 domain, Protein L mutant 1, Protein L mutant 3-Protein L mutant 8 all exhibited improved alkali resistance, and their loading capacity reached over 20 mg / ml, demonstrating good IgG (Kappa light chain only) capture ability. Compared to the control group (Cytiva product, B3 domain mutation), all mutants showed a higher 10% dynamic loading capacity, reflecting better IgG (Kappa light chain only) capture ability. Furthermore, the residual capacity of Protein L mutant 1, Protein L mutant 3-Protein L mutant 8 after 50 cycles of alkali resistance showed stronger alkali resistance than the control group.

[0165] The above are merely some preferred embodiments of the present invention, and the present invention is not limited to the contents of these embodiments. For those skilled in the art, various changes and modifications can be made within the scope of the present invention's technical solutions, and any such changes and modifications are within the protection scope of the present invention.

Claims

1. A recombinant Protein L protein, a mutant of Protein L protein domain B5, characterized in that: The amino acid sequence of the recombinant Protein L is selected from any one of the amino acid sequences shown in SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO:

42.

2. A polynucleotide encoding the recombinant Protein L protein of claim 1.

3. A vector comprising the polynucleotide of claim 2.

4. A transformant comprising the carrier as described in claim 3.

5. A chromatography medium comprising a solid support and the recombinant Protein L protein of claim 1 bound to the solid support.

6. The application of the chromatography medium of claim 5 for separating antibodies or fragments thereof containing a κ light chain.

7. A method for preparing recombinant Protein L protein, comprising expressing the recombinant Protein L protein of claim 1 in the transformant or cell-free protein synthesis system of claim 4, or by chemical synthesis.

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