Recombinant protein l protein with improved alkali resistance and use thereof

Abstract

The present application relates to the technical field of protein engineering, and specifically provides a recombinant Protein L protein with improved alkali resistance and use thereof. The recombinant Protein L protein of the present application has higher stability under alkaline conditions while maintaining high binding ability to immunoglobulins, thereby providing a more efficient and economical method for isolating immunoglobulins.

Description

Recombinant protein l protein with improved alkali resistance and application thereof

[0001] Cross-reference to related applications

[0002] The present application claims priority to the Chinese patent application No. 202411403687.3 filed on October 09, 2024, and titled "Recombinant Protein L protein with improved alkali resistance and application thereof", the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0003] The present application relates to protein engineering technology, in particular to a recombinant Protein L protein with improved alkali resistance, a preparation method and application thereof. BACKGROUND

[0004] Antibody purification is a key step in the field of biopharmaceuticals, directly affecting the quality and efficacy of antibody drugs. In the process of antibody purification, affinity chromatography with ligands with antibody binding ability is widely used. As ligands that specifically bind to antibodies, protein A, protein L, protein G and other antibody binding domains can be used.

[0005] Among them, protein L derived from the cell surface of Finegoldia magna can specifically bind to the kappa light chain of antibodies, especially K1, K3 and K4 subtypes. Compared with other antibody binding proteins such as Protein A and Protein G, Protein L has a wider binding range of Ig and Ig subtypes, and can bind to all Ig classes (IgG, IgM, IgA, IgE and IgD); In addition, it can also bind to single-chain antibodies and Fab fragments. These antibody fragments have better tissue penetration ability and lower immunogenicity due to their smaller molecular size, so they have more obvious advantages in some therapeutic applications. This unique binding property enables protein L to effectively capture antibodies and their fragments from a variety of biological samples, so the performance of protein L has attracted much attention in the purification of antibody fragment drugs.

[0006] The ligand of the conventional Protein L affinity chromatography medium on the market is natural Protein L, which cannot withstand in-situ cleaning with high-concentration NaOH and other alkali solutions during the purification process. Under strong alkaline conditions, protein L will lose stability, leading to antibody aggregation or loss of activity; resulting in short service life, low binding capacity and low purification efficiency of the affinity chromatography column. Therefore, the development of alkali-resistant media is crucial for improving the cleaning efficiency of the purification process and reducing production costs, and researchers have been exploring ways to improve the alkali stability of the medium.

[0007] There have been studies on genetically engineering Protein L to improve its stability under high pH conditions. Since S. digestivum is divided into 3316 and 312 strains, the natural protein L is composed of four (3316) or five (312) highly homologous domains C1-C4 or B1-B5, respectively, depending on the isolated bacterial strain, each domain consisting of 72-76 amino acids, and the sequence similarity between them can reach 70-80%. Protagenava Co., Ltd. (CN108291220B) from Japan performed a combination of specific amino acid substitutions on protein L from 3316 strain, which can tolerate 100 mM NaOH alkaline solution treatment. Considering the high sequence homology between 3316 and 312 strains of protein L, the same amino acid combination substitution was performed on protein L from 312 strain, but the same alkali stability improvement effect was not achieved, so this combination of variations is specific to 3316 protein L. Boguon Biotechnology Co., Ltd. (CN116675747A) from China selected protein L from 312 strain and designed only the B5 domain to obtain protein L that can tolerate 50 mM NaOH treatment, but in the production process of antibodies, the alkali treatment concentration must be at least 100 mM or more to achieve complete cleaning effect, thereby ensuring the purification efficiency. In addition, there are studies comparing the alkali stability of individual natural domains (2017, Cytiva), which found that the single B3 domain has relatively optimal alkali stability, but it is still far from the required effect in application scenarios. Therefore, for protein L from 312 strain, how to change to improve the stability under high concentration of alkali solution has not yet been clearly concluded.

[0008] In view of this, the present application is proposed.

[0009] SUMMARY

[0010] To solve the above technical problems, the present application directly selects a single domain in the sequence of protein L from 312 strain as the research object, uses structure simulation, rational design and protein stability prediction algorithm combination to further design, and finally provides a protein L mutant protein with significantly improved alkali stability.

[0011] Specifically, the present application provides the following technical solutions:

[0012] The present application first provides a recombinant Protein L protein with improved alkali resistance, which includes at least three amino acid site changes in the sequence of wild Protein L protein;

[0013] Preferably, the protein comprises at least three amino acid position changes in the sequence of the B3 Domain or B5 Domain of wild-type Protein L protein.

[0014] The present application also provides a method for improving the alkaline tolerance of Protein L protein, which comprises introducing at least three amino acid position changes in the sequence of wild-type Protein L protein.

[0015] Preferably, the method comprises introducing at least three amino acid position changes in the sequence of the B3 Domain or B5 Domain of wild-type Protein L protein.

[0016] In some aspects, the positions comprise positions 10, 45 and 60 of the B3 Domain.

[0017] In some specific aspects, the changes comprise N10Y / K, N45Y / H and N60Y / R of the B3 Domain.

[0018] In other aspects, the positions comprise positions 18, 59 and 68 of the B5 Domain.

[0019] In other specific aspects, the changes comprise N18V / F, D59I / Y and N68Y of the B5 Domain.

[0020] In some embodiments, the sequence of the B3 Domain is as set forth in SEQ ID NO. 2 and the sequence of the B5 Domain is as set forth in SEQ ID NO. 3.

[0021] In other embodiments, the sequence of the wild-type Protein L protein is as set forth in SEQ ID NO. 1.

[0022] In some specific embodiments, the sequence of the recombinant Protein L protein with improved alkaline tolerance is as set forth in SEQ ID NO. 4-8, or has more than 85% homology with SEQ ID NO. 4-8; preferably, the homology is derived from conservative substitution of amino acids.

[0023] In some preferred specific embodiments, the sequence of the recombinant Protein L protein with improved alkaline tolerance is as set forth in SEQ ID NO. 9-11, or has more than 85% homology with SEQ ID NO. 9-11; preferably, the homology is derived from conservative substitution of amino acids.

[0024] Further, the recombinant Protein L protein is in a tetramer form, specifically a tetramer form corresponding to the B3 Domain, or a tetramer form corresponding to the B5 Domain.

[0025] Further preferably, the sequence of the tetramer is shown in SEQ ID NO. 12-19, or has more than 85% homology with SEQ ID NO. 12-19; preferably, the homology is derived from conservative substitution of amino acids.

[0026] Further preferably, the sequence of the tetramer is shown in SEQ ID NO. 12-16, or has more than 85% homology with SEQ ID NO. 12-16; preferably, the homology is derived from conservative substitution of amino acids.

[0027] Further, the recombinant Protein L protein can tolerate an alkali concentration of up to 100 mM, and maintain high binding capacity to immunoglobulin, especially to immunoglobulin containing kappa light chain.

[0028] The present application also provides a gene capable of encoding the recombinant Protein L protein as described above.

[0029] The present application also provides an expression frame comprising the gene encoding the recombinant Protein L protein as described above.

[0030] The present application also provides a plasmid comprising the gene or expression frame encoding the recombinant Protein L protein as described above.

[0031] The present application also provides a vector comprising the gene, expression frame or plasmid encoding the recombinant Protein L protein as described above.

[0032] The present application also provides a host cell comprising the gene, expression frame, plasmid or vector encoding the recombinant Protein L protein as described above.

[0033] The present application also provides an engineered microorganism comprising the gene, expression frame, plasmid, vector or host cell encoding the recombinant Protein L protein as described above.

[0034] The present application also provides a method for preparing the recombinant Protein L protein, which utilizes the expression frame, plasmid, vector, host cell or engineered microorganism as described above to obtain by expression; or to obtain by biosynthesis.

[0035] The present application also provides the use of the recombinant Protein L protein as described above in antibody separation, purification or preparation.

[0036] The application also provides a method for separating, purifying or preparing an antibody, which comprises the step of treating the antibody with the aforementioned recombinant Protein L protein.

[0037] Further, the treatment is performed at an alkali concentration of up to 100 mM.

[0038] Still further, the antibody is an immunoglobulin containing kappa light chain.

[0039] The beneficial technical effects of the application are as follows:

[0040] The application designs and prepares a series of recombinant protein L mutants, which can tolerate higher concentration and longer time of alkali environment treatment, while maintaining high binding capacity to immunoglobulin containing kappa light chain, as compared with wild-type Protein L. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 is a SDS-PAGE result diagram of the Protein L of the application after alkali treatment in 100 mM NaOH for 0 h, 8 h, 16 h and 24 h, respectively; wherein, A-H wells represent mutants pL-01 to pL-08, respectively, and I well represents wild type WT.

[0042] Figure 2 is a BLI analysis result diagram of the Protein L of the application after pre-incubation in 100 mM NaOH for 8 h, 16 h and 24 h, respectively, and then binding IgG.

[0043] Figure 3 is a SPR analysis result diagram of the Protein L of the application after alkali treatment in 100 mM NaOH for 10 min*100 cycles.

[0044] DETAILED DESCRIPTION

[0045] The application discloses a recombinant protein L protein with improved alkali stability, while maintaining high binding capacity to immunoglobulin containing kappa light chain. Those skilled in the art can refer to the content herein to realize its application. It is particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the application. The preparation method and application of the application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the preparation method and application herein without departing from the content, spirit and scope of the application, to realize and apply the technology of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art.

[0046] The following terms or definitions are provided merely to aid in the understanding of the present application. These definitions should not be construed to have a narrower scope than the terms allow under ordinary and customary practices based on the preambles actually used in the various claims.

[0047] Unless otherwise defined, all technical and scientific terms used in the present application are intended to have the same meaning as commonly understood by one of ordinary skill in the art. Although the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the present application.

[0048] As used in the present application, the terms "comprising", "containing", "having" "including" or "involving" are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. The term "consisting of is considered a preferred embodiment of the term "comprising". If a group of items is defined in the alternative, such as "at least one of A or B", it is to be understood that this includes the group consisting of only A or consisting of only B.

[0049] The indefinite article "a" or "an" or the definite article "the" as used in reference to a singular noun form also includes the plural form of that noun unless the context clearly indicates otherwise.

[0050] The terms "about", "approximately" in the present application mean an interval of accuracy that a person of skill in the art would understand as still guaranteeing the technical effect of the feature in question. This term generally indicates a ±10% deviation from the indicated numerical value, preferably ±5%.

[0051] The terms "or more", "at least", "exceeding" and the like, such as "at least one", are to be understood as including, but not limited to, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more than the stated value. Any larger number or fraction in between is also included.

[0052] Conversely, the term "no more than" includes every value less than the stated value. For example, "no more than 100 nucleotides" includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, and 0 nucleotides. Any smaller number or fraction in between is also included.

[0053] The terms "a plurality," "at least two," "two or more," "at least a second," and the like, should be understood to include but not be limited to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more. Any larger number or fraction in between is also included.

[0054] Detailed description of the variant proteins of the present application

[0055] The recombinant Protein L protein with improved alkaline resistance of the present application comprises at least three amino acid site changes in the sequence of wild-type Protein L protein; particularly, at least three amino acid site changes in the sequence of B3 Domain or B5 Domain of wild-type Protein L protein. The recombinant Protein L protein can tolerate an alkaline concentration of up to 100 mM and maintain high binding capacity to kappa light chain immunoglobulin. In some aspects, the sites include positions 10, 45 and 60 of B3 Domain; in some specific aspects, the changes include N10Y / K, N45Y / H and N60Y / R of B3 Domain. In other aspects, the sites include positions 18, 59 and 68 of B5 Domain; in other specific aspects, the changes include N18V / F, D59I / Y and N68Y of B5 Domain.

[0056] It can be understood that "variant" in the text is just a form of expression, and any sequence comprising three amino acid site changes in the sequence of B3 Domain or B5 Domain of wild-type Protein L protein falls within the scope of "variant sequence" described herein, so the variant can comprise a mutant form, as well as a non-mutant form. In addition, the specific source form or preparation means of the variant are not limited, for example, in some specific embodiments, the variant is directly obtained by chemical synthesis; in other specific embodiments, the variant is directly obtained by recombinant expression; in other specific embodiments, the variant can be obtained by introducing mutant amino acids into the wild-type sequence by genetic engineering or genetic means.

[0057] In addition, "change" in the text includes mutant form, but is not limited to mutation. It can be understood that the variant sequence obtained by non-mutant form also has the same effect and function in the present application, which also falls within the reasonable protection scope of the present application.

[0058] In general, the wild-type Protein L protein as used herein can have at least about 85% sequence identity, more particularly at least about 85%, 90%, 95% or 99% sequence identity to the protein of SEQ ID NO. 1. In some embodiments, the wild-type Protein L protein has an amino acid sequence as specifically set forth in SEQ ID NO. 1. It is understood, however, that Protein L proteins obtained from different sources or different habitats can have individual amino acid differences in the wild-type sequence, but they are also understood to be wild-type sequences, and thus the wild-type Protein L protein amino acid sequence of the present application should not be limited in theory to the sequence of SEQ ID NO. 1, and wild-type sequences having at least about 85% sequence identity, more particularly at least about 90%, 95% or 99% sequence identity to SEQ ID NO. 1 are within the scope of the present application, provided that the core functional Domain sequences are consistent, and provided that the mutations described herein made on the basis of these underlying wild-type sequences are also within the scope of the present application.

[0059] SEQ ID NO. 1 : Wild-type Protein L protein

[0060] In some embodiments of the present application, the sequence of the B3 Domain is as set forth in SEQ ID NO. 2, and the sequence of the B5 Domain is as set forth in SEQ ID NO. 3. Again, it is understood that Protein L proteins obtained from different sources or different habitats can have individual amino acid differences in the wild-type sequence of the B3 or B5 Domain (such as non-functional amino acids, particularly conservative changes), and these differences also retain the original function, and thus the mutations described herein made on the basis of these underlying B3 or B5 sequences are also within the scope of the present application, and should not be limited to SEQ ID NO. 2 or 3.

[0061] SEQ ID NO. 2: B3 Domain

[0062] SEQ ID NO. 3: B5 Domain

[0063] In some specific embodiments of the present application, the mutant functional domain sequence of the recombinant Protein L protein having improved alkaline resistance is as specifically set forth in SEQ ID NO. 4-11.

[0064] SEQ ID NO. 4: (B3 domain, N10Y, N45Y, N60Y) SEQ ID NO. 4: (B3 domain, N10Y, N45Y, N60Y)

[0065] SEQ ID NO. 5: (B3 domain, N10Y, N45Y, N60R)

[0066] SEQ ID NO. 6: (B3 domain, N10Y, N45H, N60R)

[0067] SEQ ID NO. 7: (B3 domain, N10K, N45Y, N60R)

[0068] SEQ ID NO. 8: (B3 domain, N10K, N45H, N60R)

[0069] SEQ ID NO. 9: (B5 domain, N18V, D59I, N68Y)

[0070] SEQ ID NO. 10: (B5 domain, N18F, D59I, N68Y)

[0071] SEQ ID NO. 11: (B5 domain, N18V, D59Y, N68Y)

[0072] In some more specific embodiments, to further improve the number of antibodies that can be bound and the alkaline stability, the recombinant protein form of the present application adopts a repeating domain structure composed of four mutated domains, specifically a tetramer form corresponding to the B3 Domain, or a tetramer form corresponding to the B5 Domain, and the specific sequences are shown in SEQ ID NO. 12-19. As can be seen from Table 2, these sequences all exhibit good alkali resistance activity and antibody binding activity, and the specific sequences are as follows:

[0073] SEQ ID NO. 12: (B3 domain, N10Y, N45Y, N60Y) tetramer

[0074] SEQ ID NO. 13: (B3 domain, N10Y, N45Y, N60R) tetramer

[0075] SEQ ID NO. 14: (B3 domain, N10Y, N45H, N60R) tetramer

[0076] SEQ ID NO. 15: (B3 domain, N10K, N45Y, N60R) tetramer

[0077] SEQ ID NO. 16: (B3 domain, N10K, N45H, N60R) tetramer

[0078] SEQ ID NO. 17: (B5 domain, N18V, D59I, N68Y) tetramer

[0079] SEQ ID NO. 18: (B5 domain, N18F, D59I, N68Y) tetramer

[0080] SEQ ID NO. 19: (B5 domain, N18V, D59Y, N68Y) tetramer

[0081] Although the present application provides the above specific sequence tetramer, it is clear in the art that the present application can also include sequences with certain homology to the above sequences, which homology is derived from conservative mutations of amino acids, such as conservative substitutions, which means that one amino acid is substituted by another amino acid within the same class, for example, one acidic amino acid is substituted by another acidic amino acid, one basic amino acid is substituted by another basic amino acid, or one neutral amino acid is substituted by another neutral amino acid. Exemplary substitutions are shown in the following table (amino acid substitutions).

[0082] Therefore, in some embodiments of the present application, the recombinant protein with improved alkaline resistance can also include at least about 85% sequence identity, in particular at least about 85%, 90%, 95% or 99% sequence identity, to any one of SEQ ID NO. 4-19.

[0083] In addition, it can also be understood that, on the basis of meeting the general binding ability of the protein, the skilled person in the art can further carry out other known effective site mutations on the basis of the present application to meet the needs of practical optimization and improvement. Such mutations, because they occur on the basis of the present application, are also within the scope of protection of the present application, and the corresponding effects are reasonably expected by the skilled person in the art on the basis of the basic understanding of the structure of the protein.

[0084] The technical solutions of the present application will be clearly and completely described below in combination with the embodiments. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application. Embodiments

[0085] The technical solutions of the present application will be clearly and completely described below in combination with the embodiments. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0086] Embodiment 1, mutant design and construction

[0087] The present application respectively selects B3 domain (SEQ ID NO. 2) and B5 domain (SEQ ID NO. 3) derived from Protein L protein of Streptococcus 312 strain as a template, and then mutates amino acids sensitive to alkaline environment, such as asparagine, to reduce the sensitivity of the mutated domain to alkaline environment.

[0088] Specifically, through previous mutation screening, protein fragments with B3 domain sequence of SEQ ID NO. 4-8 and B5 domain of SEQ ID NO. 9-11 are obtained; in order to further improve the number of antibodies that can be combined and the alkaline stability, the final mutant construct of the present application is further constructed to consist of four mutated repeat domains, and the sequences of the four mutant tetramer obtained are shown as follows: SEQ ID NO. 12-19. After the corresponding gene fragments are synthesized by General Biosystems Co., Ltd., they are respectively inserted into a cell-free expression vector to obtain a recombinant expression plasmid template. The specific mutant representation is shown in Table 1.

[0089] Table 1

[0090] Embodiment 2, expression and purification of recombinant mutants

[0091] The present application selects an E. coli cell-free synthesis system for mutant expression. The plasmid template in Example 1 is added to the cell-free reaction system at a ratio of 1:250, which contains BL21(DE3) cell extract, amino acids, energy sources, salts and other auxiliary factors, etc. After mixing uniformly, it is placed at 30°C and cultured at a speed of 250 rpm for 16 h. Then the culture supernatant is collected by centrifugation at a speed of 12,000 rpm for 20 min, and filtered using a 0.45 μm filter membrane. The filtered sample is loaded onto a nickel column NiFF, and eluted using PBS buffer containing 30 mM / 100 mM / 500 mM imidazole, respectively. The target protein is eluted at a concentration of 100 mM imidazole, and the protein purity is greater than 90% detected by SDS-PAGE. Then the protein concentration is measured by spectrophotometry at a wavelength of 280 nm.

[0092] Example 3, Comparison experiment of protein alkali resistance of different mutants

[0093] In order to determine the improvement of the alkali resistance of the mutated protein L, the natural wild type and all mutant proteins were incubated in 100 mM NaOH at room temperature for 0 h, 8 h, 16 h and 24 h, respectively, and then neutralized to neutral conditions. The following evaluations were performed:

[0094] 1. SDS-PAGE degradation detection to observe whether the protein is broken or degraded. The detection results are shown in Figure 1. The degradation trend of the wild type protein becomes more and more obvious with the increase of alkali treatment time (I lane); the mutant proteins pL-06 to pL-08 show slight degradation after 24 h of alkali treatment; and the mutant proteins pL-01 to pL-05 do not show obvious breakage or degradation, indicating that their alkali resistance has been significantly improved compared with the wild type.

[0095] 2. All mutants and wild type were arranged for intermolecular interaction analysis. The interaction between each mutant protein and human IgG was analyzed using the molecular interaction device ForteBio Octet 96e. Biotin-labeled human IgG (Trastuzumab) was captured by SA Biosensor as an IgG immobilized chip. The protein L protein after alkali incubation for different times was diluted to 50 nM using PBS buffer containing 0.1% BSA, and was combined and dissociated with the chip as an analyte. For a single mutant protein L, the residual IgG binding capacity (%) was characterized by comparing the binding signal value of different alkali incubation times with the binding signal value before alkali treatment.

[0096] The analysis results are shown in Table 2 and Figure 2. Mutants pL-01 to pL-05 can still maintain more than 70% of IgG binding capacity after 24h of alkaline incubation, while the wild-type protein only remains 48% of the binding capacity.

[0097] Table 2

[0098] Note: The result of pl-01 exceeds 100% because the BLI detection system has normal fluctuations. In practice, a measurement error of 10% or more is considered normal. The detection of 108.0% in this case at least indicates that pl-01 has very high binding capacity.

[0099] Example 3, Comparison of the Alkali Resistance of Different Mutants

[0100] The above-mentioned mutants with strong remaining IgG binding capacity were used to confirm again using a Biacore 8K instrument. Wild-type and mutant protein L proteins (both not incubated with 100mM NaOH) were immobilized on a Biacore CM5 sensor chip using an amine coupling kit (the immobilization signal intensity was about 1500RU). Then 30nM IgG was used to flow through the chip and the signal intensity after 60s was recorded. In order to characterize the resistance to alkaline environment, after the IgG flowed through the chip, in-situ cleaning was performed using 100mM NaOH solution at room temperature at a speed of 20μl / min for 10min, repeating 100 cycles. The average RU signal intensity of every 10 cycles was taken as the signal value of the current 10 cleaning cycles, and the average value of the previous 10 cycles was taken as 100%. The alkaline stability of the immobilized protein L ligand was represented by the relative loss of signal intensity.

[0101] The results are shown in Figure 3. Compared with the wild-type, mutants pL-01 to pL-05 have significantly enhanced alkaline stability, and can still maintain 85% of the remaining binding capacity after 100 cycles of alkaline cleaning, which can significantly reduce the cost and improve the efficiency of antibody purification.

[0102] The results of the above examples show that simultaneous mutation of multiple amino acids based on the sequences of B3 Domain or B5 Domain of Protein L protein can effectively increase the alkali resistance.

[0103] The foregoing description of specific exemplary embodiments of the application has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the application to the precise form disclosed, and various modifications and variations are possible in light of the above teachings. It is intended that the embodiments be limited only by the claims as interpreted in their broadest terms.

Claims

1. A recombinant Protein L protein with improved resistance to alkali, characterized in that, The protein comprises at least three amino acid site changes in the sequence of B3 Domain or B5 Domain of wild-type Protein L protein.

2. A method for improving the alkali resistance of Protein L protein, characterized by, The protein comprises at least three amino acid site changes in the sequence of B3 Domain or B5 Domain of wild-type Protein L protein.

3. The recombinant Protein L protein of claim 1 or the method of claim 2, wherein, The sites comprise 10th, 45th and 60th sites of B3 Domain, or 18th, 59th and 68th sites of B5 Domain; preferably, the changes comprise N10Y / K, N45Y / H and N60Y / R of B3 Domain, or N18V / F, D59I / Y and N68Y of B5 Domain.

4. The method of claim 1 or 2 or 3, characterized in that, The sequence of B3 Domain is shown in SEQ ID NO. 2 or has more than 85% homology with SEQ ID NO. 2; the sequence of B5 Domain is shown in SEQ ID NO. 3 or has more than 85% homology with SEQ ID NO. 2; preferably, the homology is derived from conservative substitution of amino acids.

5. The method of claim 1 or 2 or 3, wherein, The sequence of the recombinant Protein L protein with improved alkali resistance is shown in SEQ ID NO. 4-11, or has more than 85% homology with SEQ ID NO. 4-11; preferably, the homology is derived from conservative substitution of amino acids.

6. The method of claim 1 or 3, wherein The recombinant Protein L protein is a B3 Domain tetramer or a B5 Domain tetramer; preferably, the sequence of the tetramer is shown in SEQ ID NO. 12-19, or has more than 85% homology with SEQ ID NO. 12-19; more preferably, the homology is derived from conservative substitution of amino acids.

7. A gene, characterized in that, The gene encodes the recombinant Protein L protein of any one of claims 1, 3-6.

8. Expression cassette, plasmid, vector, host cell or engineered microorganism, characterized in that, The gene of claim 7.

9. A method for preparing a recombinant Protein L protein, characterized by using the expression frame, plasmid, vector, host cell or engineered microorganism of claim 8 to express.

10. Use of the recombinant Protein L protein of any one of claims 1, 3-6 in antibody separation, purification or preparation.

11. A method of isolating, purifying or preparing an antibody, characterized in that, The method comprises the step of treating the antibody with the recombinant Protein L protein of any one of claims 1, 3-6; preferably, the treatment is performed at an alkali concentration of up to 100 mM.

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