Proteins having affinity for immunoglobulins and affinity separation agents, liquid chromatography columns using the same

Abstract

The present application provides a protein having resistance to serine protease or thermolysin and immunoglobulin binding activity, which can be used as an affinity ligand. One embodiment of the present application is a protein having affinity to immunoglobulin, having two or more domains derived from any one of the E, D, and A domains of protein A, containing one or more lysines in at least one amino acid sequence of the domain, and having a lysine deleted or replaced at the C-terminus; or a protein having affinity to immunoglobulin, having two or more domains derived from any one of the B, C, and Z domains of protein A, containing one or more lysines in at least one amino acid sequence of the domain, and having a lysine deleted or replaced at the 4th position and at the C-terminus.

Description

Technical Field

[0001] This invention relates to proteins that specifically bind to immunoglobulins, affinity separating agents that use the proteins as immunoglobulin-binding affinity ligands, and liquid chromatography columns. Background Technology

[0002] Antibodies have the function of specifically binding to substances called antigens and working in conjunction with other biological molecules and cells to detoxify and remove antigenic factors. The name "antibody" emphasizes this function of binding to antigens; as a substance, it is called "immunoglobulin".

[0003] In recent years, with the development of genetic engineering, protein engineering, and cell engineering, the development of pharmaceuticals utilizing the functions of antibodies, known as antibody drugs, has become increasingly popular. Because antibody drugs act more specifically on target molecules compared to existing drugs, they are expected to have fewer side effects and achieve better therapeutic effects, actually contributing to the improvement of various diseases.

[0004] On the other hand, since antibody drugs are administered to organisms in large quantities, their purity has a greater impact on quality compared to other recombinant protein drugs. Therefore, to manufacture high-purity antibodies, methods such as affinity chromatography using adsorbent materials with molecules that specifically bind to the antibody as ligands are generally employed.

[0005] Antibody drugs are primarily developed using monoclonal IgG antibodies, which are mass-produced using recombinant cell culture technology and purified using proteins that have an affinity for IgG antibodies.

[0006] Protein A is a well-known immunoglobulin-binding protein with affinity for IgG antibodies. Protein A is a cell wall protein produced by the Gram-positive bacterium *Staphylococcus aureus*, and consists of a signal sequence S, five immunoglobulin-binding domains (E, D, A, B, and C domains), and a cell wall-binding domain, the XM region (Non-Patent Literature 1). In the initial purification step (capture step) of antibody drug manufacturing, affinity chromatography columns obtained by immobilizing protein A as a ligand on a water-insoluble support are generally used.

[0007] In recent years, various developments have been carried out in connection with protein A as a ligand in order to improve the performance of this affinity chromatography column, and attempts have been made to use protein-engineered modified recombinant protein A.

[0008] For example, as recombinant protein A, there are known proteins such as protein A (rProtein ASepharose (registered trademark), manufactured by GE HealthCare Japan) with the XM region having been removed and which has no immunoglobulin binding activity, and protein A disclosed in Patent Document 1, which has a modified domain, namely the Z domain, obtained by introducing a mutation into the B domain.

[0009] The Z domain is a modified domain derived from the B domain by replacing the glycine at position 29 with alanine. Compared to the B domain, it exhibits higher alkali resistance. Therefore, to improve ligand function, recombinant protein A with enhanced alkali resistance was developed, enabling antibodies to dissolve in weakly acidic conditions.

[0010] In Patent Document 2, in order to improve alkali resistance, an affinity chromatographic ligand containing the N-terminus of 3 to 5 consecutive amino acids of the C, B, and Z domains of protein A is disclosed.

[0011] In Patent Document 3, in order to improve alkali stability, an affinity chromatographic ligand having an Asn-Lys-Phe-Asn deletion sequence at positions 3 to 6 of the C unit of protein A was disclosed.

[0012] In patent document 4, in order to improve alkali resistance, an immunoglobulin-binding protein was disclosed in which the asparagine at positions 3 and 6 of the B and Z domains of protein A was replaced with other amino acids.

[0013] In Patent Document 5, in order to enable the antibody to detach under weak acidity, an antibody recognition binding protein is disclosed that does not contain lysine and cysteine ​​residues in the E, D, A, B, and C domains of protein A.

[0014] Existing technical documents

[0015] Patent documents

[0016] Patent Document 1: US Patent No. 5,143,844

[0017] Patent Document 2: Japanese Patent Application Publication No. 2012-254981

[0018] Patent Document 3: Japanese Patent Publication No. 2010-504754

[0019] Patent Document 4: Japanese Patent Publication No. 2005-538693

[0020] Patent Document 5: Japanese Patent Application Publication No. 2013-256484

[0021] Non-patent literature

[0022] Non-patent literature 1: Hober S. et al., J. Chromatogr. B., 2007, Vol. 848, pp. 40-47 Summary of the Invention

[0023] Through the research of the inventors, the following problem was discovered for the first time: due to the action of the protease produced by the host microorganism used in the production of recombinant protein A or the protease produced by the cultured cells during the purification of IgG antibodies produced by recombinant cultured cells, protein A, as a ligand, is cleaved, and the immunoglobulin binding activity is greatly reduced.

[0024] It has been identified that protease-induced cleavage is a significant problem, particularly during the purification of recombinant protein A from culture medium by raising the pH to allow it to adsorb or flow through an ion exchange resin, during concentration or desalting processes before or after purification, and during the purification of immunoglobulins from culture medium containing immunoglobulins using a separating agent immobilized with recombinant protein A.

[0025] As one of the aforementioned proteases, there is a serine protease. The recombinant protein A disclosed in Patent Documents 1-4 lacks resistance to serine proteases, posing a problem when used as a ligand for affinity chromatography columns.

[0026] In addition, according to the research of the inventors, the recombinant protein A disclosed in Patent Document 5, which does not contain lysine and cysteine, has the following problems: insufficient immunoglobulin binding activity, especially when polymerized, the immunoglobulin binding activity is low compared with the wild type, and ultimately it cannot fully perform its function as a ligand for affinity chromatography columns.

[0027] In addition, another type of protease that causes the aforementioned problems is thermophilic protease. Thermophilic proteases are metalloproteinases derived from Bacillus bacteria, exhibiting high activity in high-temperature and high-pH environments. Since the recombinant protein A disclosed in Patent Documents 1, 3-5 lacks resistance to thermophilic proteases, it is impossible to recover the protein in its intact form, posing a problem when used as a ligand for affinity chromatography columns.

[0028] Furthermore, according to the research of the inventors, a new problem has been discovered: although the recombinant protein A disclosed in Patent Document 2 contains a protein resistant to thermophilic proteases, it suffers from reduced immunoglobulin binding activity and ultimately cannot fully perform its function as a ligand for affinity chromatography columns.

[0029] Therefore, the objective of this invention is to provide a protein that is resistant to proteases and has sufficient immunoglobulin binding activity, suitable for use as an affinity ligand.

[0030] In particular, the first objective of this invention is to provide a protein that is resistant to proteases, especially serine proteases, and has sufficient immunoglobulin binding activity, and is suitable for use as an affinity ligand.

[0031] Furthermore, a second objective of the present invention is to provide a protein that is resistant to proteases, particularly thermophilic proteases, and has sufficient immunoglobulin binding activity, making it suitable for use as an affinity ligand.

[0032] In order to solve the first problem mentioned above, the inventors conducted in-depth research and found that when the region connecting the domains in the amino acid sequence of a specific domain of protein A does not contain lysine, the resistance to serine proteases is improved, thus completing the present invention.

[0033] In addition, the inventors conducted in-depth research to solve the second problem mentioned above, and found that when the region connecting the domains in the amino acid sequence of a specific domain of protein A does not contain hydrophobic amino acids, the resistance of thermophilic bacteria to proteases is improved, thus completing the present invention.

[0034] That is, the above-mentioned problem is achieved through the following structure.

[0035] [1] A protein having affinity for immunoglobulins, characterized in that it has two or more domains derived from any one of the E, D and A domains of protein A described in sequence numbers 1 to 3, wherein at least one amino acid sequence of the domain contains one or more lysine residues and the C-terminal lysine residue is missing or replaced.

[0036] [2]-(1) A protein having affinity for immunoglobulins, characterized in that it has two or more domains derived from any one of the B, C and Z domains of protein A described in sequence numbers 4 to 6, wherein at least one amino acid sequence of the domain contains one or more lysines and the lysine at position 4 and the lysine at the C-terminus is missing or replaced.

[0037] [2]-(2) A protein with affinity for immunoglobulins, characterized in that it has two or more domains derived from any one of the B, C and Z domains of protein A described in sequence numbers 4 to 6, wherein at least one amino acid sequence of the domain contains one or more lysine residues, and when the C-terminal lysine residue of each domain and the sequence of the first to fifth positions of the amino acid sequence of each domain are used as linker sequences (first linker elements), at least one C-terminal lysine residue and the lysine residue at the fourth position of the linker sequence (first linker element) are deleted or replaced.

[0038] [3] The protein according to [1] or [2], wherein, of the lysine at position 4 and / or the lysine at position C-terminus that are missing or replaced, the lysine at position 4 and / or the lysine at position C-terminus are missing.

[0039] [4] The protein according to any one of [1] to [3], wherein when the lysine residue at the C-terminus of each domain and the sequence at position 1 to position 5 of the amino acid sequence of each domain are used as the linking position sequence (first linking element), at least one of the linking position sequences (first linking element) consists of one or more amino acids.

[0040] [5] The protein according to any one of [1] to [4], wherein the lysine at the fourth position and / or the lysine at the C-terminus, which are missing or replaced, are replaced with hydrophilic amino acids.

[0041] [6] The protein according to any one of [1] to [5], wherein it has three or more domains.

[0042] [7] The protein according to any one of [1] to [6], wherein when the lysine at the C-terminus of each domain and the sequence at position 1 to position 5 of the amino acid sequence of each domain are used as the linker sequence (first linker element), lysine is missing or replaced in all the linker sequences (first linker elements).

[0043] [8] The protein according to any one of [1] to [7], wherein all the domains contained in the protein are derived from any one of the amino acid sequences of the E, D, A, B, C and Z domains of protein A recorded in sequence numbers 1 to 6.

[0044] [9] The protein according to any one of [1] to [8], wherein when the lysine residue at the C-terminus of each domain and the sequence at position 1 to position 5 of the amino acid sequence of each domain are used as the linking position sequence (first linking element), the linking position sequence (first linking element) does not contain lysine residue.

[0045]

[10] The protein according to any one of [1] to [9], wherein when the lysine at the C-terminus of each domain and the sequence at position 1 to position 5 of the amino acid sequence of each domain are used as the linking position sequence (first linking element), the linking position sequence (first linking element) does not contain arginine.

[0046]

[11] An affinity separating agent, characterized in that the protein described in any one of [1] to

[10] is used as an affinity ligand and is immobilized on a support made of a water-insoluble substrate.

[0047]

[12] A liquid chromatography column, characterized in that it comprises the affinity separating agent described in

[11] and has at least one container.

[0048]

[13] A protein having affinity for immunoglobulins, characterized in that it has two or more domains derived from any one of the A, B, C and Z domains of protein A described in sequence numbers 3 to 6, wherein when the sequence of the first to fifth position of the amino acid sequence of each domain is used as a linker (second linker), at least one linker (second linker) is a variant linker (variant second linker) composed of one or more amino acids and composed of amino acids other than hydrophobic amino acids.

[0049]

[14] According to the protein described in

[13] , wherein the above-mentioned variant linker element (variant second linker element) is composed of the sequence of the first to fifth positions of the amino acid sequence of the A, B or C domain, with the alanine at the first position and / or the phenylalanine at the fifth position missing.

[0050]

[15] According to the protein described in

[13] , wherein the above-mentioned variant linker element (variant second linker element) is composed of the sequence of the first to fifth positions of the amino acid sequence of the Z domain, with the first position of valine and / or the fifth position of phenylalanine missing.

[0051]

[16] According to the protein described in

[13] or

[14] , wherein the above-mentioned variant linker element (variant second linker element) is composed of the sequence of the first to fifth positions of the amino acid sequence of the A, B or C domain, and the alanine at the first position and / or the phenylalanine at the fifth position are replaced by an amino acid other than a hydrophobic amino acid.

[0052]

[17] According to the protein described in

[13] or

[15] , wherein the above-mentioned variant linker element (variant second linker element) is composed of the sequence of the first to fifth positions of the amino acid sequence of the Z domain, wherein the valine at the first position and / or the phenylalanine at the fifth position are replaced by an amino acid other than a hydrophobic amino acid.

[0053]

[18] The protein according to

[16] or

[17] , wherein the amino acid other than the hydrophobic amino acid replaced in the above-mentioned variant linker element (variant second linker element) is a hydrophilic amino acid.

[0054]

[19] The protein according to any one of

[13] to

[18] , wherein the above-mentioned variant linking element (variant second linking element) is composed of two or more amino acids.

[0055]

[20] The protein according to any one of

[13] to

[19] , wherein it has three or more domains.

[0056]

[21] The protein according to any one of

[13] to

[20] , wherein all of the linking elements (second linking elements) are variant linking elements (variant second linking elements).

[0057]

[22] The protein according to any one of

[13] to

[21] , wherein all the domains contained in the protein are derived from any one of the amino acid sequences of the A, B, C and Z domains of protein A recorded in sequence numbers 3 to 6.

[0058]

[23] The protein according to any one of

[13] to

[22] , wherein a C-terminal lysine residue in an amino acid sequence of at least one domain is missing or replaced.

[0059]

[24] The protein according to

[23] wherein, in the domain on the C-terminus side that binds the above-mentioned variant linker element (variant second linker element), the C-terminal lysine in the amino acid sequence of the domain is missing or replaced.

[0060]

[25] The protein according to any one of

[13] to

[24] , wherein the above-mentioned variant linker element (variant second linker element) does not contain lysine.

[0061]

[26] The protein according to any one of

[13] to

[25] , wherein the above-mentioned variant linker element (variant second linker element) does not contain arginine.

[0062] The method for producing the protein described in any one of

[27]

[13] to

[26] involves transforming a Bacillus brevis bacterium with a vector containing DNA encoding the protein described in any one of

[13] to

[26] and culturing it, thereby producing the protein.

[0063]

[28] An affinity separating agent, characterized in that the protein described in any one of

[13] to

[26] is used as an affinity ligand and is immobilized on a support made of a water-insoluble substrate.

[0064]

[29] A liquid chromatography column, characterized in that it comprises the affinity separating agent described in

[28] and has at least one container filled with the affinity separating agent.

[0065] According to embodiments of the present invention, proteins that are resistant to specific proteases and possess sufficient immunoglobulin-binding activity can be provided. By using such proteins of the present invention, affinity ligands with excellent immunoglobulin-binding activity and affinity separating agents with excellent durability can be manufactured.

[0066] In particular, according to the first embodiment of the present invention, a protein that is resistant to proteases, especially serine proteases, and has sufficient immunoglobulin binding activity can be provided. By using such a protein of the present invention, affinity ligands with excellent immunoglobulin binding activity and affinity separating agents with excellent durability can be manufactured.

[0067] Furthermore, according to a second embodiment of the present invention, a protein that is resistant to proteases, particularly thermophilic proteases, and has the ability to bind to immunoglobulins can be provided. By using such a protein of the present invention, it is possible to manufacture affinity ligands with excellent ability to bind to immunoglobulins and affinity separating agents with excellent durability. Attached Figure Description

[0068] Figure 1 This is a comparative table of the amino acid sequences of the E, D, A, B, C, and Z domains of protein A from Staphylococcus. It should be noted that "- (hyphen)" indicates an amino acid residue identical to that in the C domain, and " / (slash)" indicates an amino acid deletion.

[0069] Figure 2 This is a schematic diagram of a tetrameric protein fixed to a support without forming a non-fixed domain.

[0070] Figure 3 (A) to (C) are schematic diagrams of a tetrameric protein fixed to a support and having more than one non-fixed domain.

[0071] Figure 4 (A) Figure 4 (B) is a schematic diagram of a tetrameric protein fixed to a support and having more than one non-fixed domain.

[0072] Figure 5 (A) Figure 5 (B) is a schematic diagram of a tetrameric protein immobilized on a support carrier and a fixation tag introduced to form a binding with the support carrier, with all structural domains being non-fixed structural domains. Detailed Implementation

[0073] The embodiments of the present invention will now be described in detail.

[0074] It should be noted that embodiments of the present invention relate to proteins that are resistant to specific proteases and have sufficient immunoglobulin binding activity.

[0075] The first embodiment of the present invention relates to a protein that is resistant to proteases, particularly serine proteases, and has sufficient immunoglobulin binding activity.

[0076] Furthermore, the second embodiment of the present invention relates to a protein that is resistant to proteases, particularly thermophilic proteases, and has the ability to bind to immunoglobulins.

[0077] It should be noted that in this specification, the term "protein" refers to all molecules having a polypeptide structure, including fragmented or peptide chains linked by peptide bonds.

[0078] In addition, a "domain" is a unit of higher-order structure of a protein, consisting of a sequence of tens to hundreds of amino acid residues, and is a unit of a protein that can perform some physicochemical or biochemical functions.

[0079] [First Embodiment of the Invention]

[0080] <Protein>

[0081] The protein of the first embodiment of the present invention is characterized in that it has two or more domains derived from any one of the E, D and A domains of protein A described in sequence numbers 1 to 3, wherein the amino acid sequence of at least one domain contains one or more lysine residues and the C-terminal lysine residue is missing or replaced.

[0082] The protein of the second aspect in the first embodiment of the present invention is characterized in that it has two or more domains derived from any one of the B, C and Z domains of protein A described in sequence numbers 4 to 6, wherein the amino acid sequence of at least one domain contains one or more lysine residues and the lysine residue at position 4 and the lysine residue at the C-terminus is missing or replaced.

[0083] The protein of the third aspect in the first embodiment of the present invention is characterized in that it has two or more domains derived from any one of the B, C and Z domains of protein A described in sequence numbers 4 to 6, and the amino acid sequence of at least one of the domains contains one or more lysine residues. When the C-terminal lysine residue of each domain and the sequence of the first to fifth positions of the amino acid sequence of each domain are used as linker sequences (first linker elements), at least one C-terminal lysine residue and the lysine residue at the fourth position of the linker sequence (first linker element) are deleted or replaced.

[0084] Hereinafter, in this specification, the proteins of the first, second and third embodiments of the present invention will be collectively referred to as the proteins of the first embodiment of the present invention.

[0085] Protein A is a protein composed of five immunoglobulin-binding domains linked together. Many microorganisms express protein A; for example, Staphylococcus aureus expresses protein A.

[0086] The E, D, A, B, C and Z domains of protein A described in sequence numbers 1 to 6 are immunoglobulin-binding proteins that can bind to regions other than the complementarity-determining region (CDR) of immunoglobulins. Each domain can bind to regions such as the Fc region, Fab region and especially the Fv region of the Fab region of immunoglobulins.

[0087] like Figure 1 As shown in the sequence comparison table, the E, D, A, B, C, and Z domains derived from protein A have highly similar amino acid sequences, exhibiting over 60% amino acid sequence homology. It should be noted that hyphens (-) indicate amino acid residues identical to those in the C domain.

[0088] "A domain derived from any one of the E, D, A, B, C, and Z domains of protein A" refers to a domain having the following amino acid sequence derived from the amino acid sequence of each domain in the wild type. As long as it can encode a protein capable of binding to the Fc region, variations other than the variant linker sequence (variant first linker element) described later can be introduced into the amino acid sequence of each domain in the wild type.

[0089] In other words, the amino acid sequence of any one of the E, D, A, B, C, and Z domains of protein A is the amino acid sequence before the introduction of the mutation (wild-type amino acid sequence), while the amino acid sequence "derived from any one of the E, D, A, B, C, and Z domains of protein A" refers to the wild-type amino acid sequence of the E, D, A, B, C, and Z domains themselves, or the wild-type amino acid sequence containing the mutation linking position sequence (mutated first linking element) described later and / or the amino acid sequence altered by partial substitution, insertion, deletion, and chemical modification of amino acids.

[0090] Here, the Z domain of protein A is obtained by introducing mutations such as A1V and G29A into the B domain. It is a domain that does not exist in the natural protein A. However, in this specification, the amino acid sequence represented by sequence number 6 is referred to as the wild-type amino acid sequence of the Z domain.

[0091] In this specification, the position of a specific amino acid in the amino acid sequence is determined by sequentially numbering the amino acid sequence with the N-terminus of the wild-type amino acid sequence as the first position. Typically, the N-terminus of each domain is taken as the first position. For example, the fourth amino acid, i.e., the fourth position, starting from the N-terminus of the B, C, and Z domains of protein A is lysine. Here, as... Figure 1As shown, the E domain is treated as a domain with the deletion of amino acid residues at positions 1 and 2, based on regions with high similarity to other domains. Additionally, as... Figure 1 As shown, the D domain is treated as a domain with the 3rd to 5th amino acid residues from the N-terminus inserted, based on the region with high similarity to other domains.

[0092] In this specification, when an amino acid is not formally named, it is represented by a commonly used abbreviation. Furthermore, regarding variations involving the substitution of amino acids, the wild-type or non-mutant amino acid is indicated before the substitution position number, and the mutated amino acid is indicated after the substitution position number. For example, the variation of replacing glycine (Gly) at position 29 with alanine (Ala) is recorded as G29A.

[0093] The protein of the first embodiment of the present invention is a multimeric protein (multidomain protein) having two or more of the above-mentioned "domains derived from any one of the E, D, A, B, C and Z domains of protein A" as single domains. Preferably, there are three or more, more preferably four or more, more preferably ten or less, more preferably eight or less, and even more preferably six or less.

[0094] These multimeric proteins can be linkers of a single immunoglobulin-binding domain, i.e., homodimers, homotrimers, or other homopolymers, or linkers of different types of immunoglobulin-binding domains, i.e., heterodimers, heterotrimers, or other heteropolymers. Preferably, the protein of the first embodiment of the present invention comprises all domains derived from any one of the E, D, A, B, C, and Z domains of protein A as described in sequences 1 to 6, which are homopolymers.

[0095] For the protein in the first embodiment of the present invention, the C-terminus and N-terminus of two or more domains are connected to form a linker sequence (first linker element). Here, in this specification, "linker sequence (first linker element)" refers to the lysine residue at the C-terminus of each domain and the sequence from position 1 to position 5 of the N-terminus to which it is connected.

[0096] In other words, the sequence of a total of 6 amino acid residues, consisting of 1 amino acid residue on the C-terminal side and 5 amino acid residues on the N-terminal side at the location where the two domains connect, is the linking position sequence (the first linker element). Here, the amino acid sequence on the N-terminal side refers to the 5 amino acid residues of this sequence based on the amino acid sequences preserved in B, C, and Z.

[0097] Therefore, as Figure 1As shown, since the E domain is a sequence obtained by deleting two amino acid residues from the N-terminus in the N-terminal side of the reference sequence, the linker position sequence (first linker element) in the case of the E domain refers to a sequence of four amino acid residues, namely one amino acid residue on the C-terminus side and three amino acid residues on the N-terminus side.

[0098] In addition, such as Figure 1 As shown, since the D domain is obtained by inserting three amino acid residues (represented by ()) into the N-terminal sequence as a baseline, the protein containing the D domain consists of a total of nine amino acid residues, including one amino acid residue at the C-terminus and eight amino acid residues at the N-terminus in the linker portion between the domains. It is considered that the three amino acid residues "AQQ" inserted at the N-terminus of the D domain do not particularly affect the effectiveness of the invention. Therefore, in this specification, based on the amino acid sequences retained in B, C, and Z, the amino acid sequence at positions 1 to 5 of the N-terminus of the D domain included in the linker position sequence (first linker element) refers to the five amino acid residues shown below.

[0099] n-mer proteins contain n-1 linker sequences (the first linker element).

[0100] like Figure 1 As shown, the C-terminus of any one of the E, D, A, B, C, and Z domains of protein A is a lysine (K). Therefore, the above-mentioned linker sequence (the first linker element) must contain a lysine (K).

[0101] Additionally, the sequences shown below are the amino acid sequences of positions 1 through 5 of the E, D, A, B, C, and Z domains of protein A. The E domain lacks amino acid residues at positions 1 and 2, consists of 3 amino acids, and does not contain lysine (K). Each of the D, A, B, C, and Z domains consists of 5 amino acids; the D and A domains do not contain lysine (K), while the B, C, and Z domains contain lysine (K) at position 4.

[0102]

[0103] The proteins of the first and third embodiments of the present invention are characterized in that at least one linker sequence (first linker element) is a variant linker sequence (variant first linker element) altered by partial amino acid substitution, insertion, deletion, and chemical modification, and is an amino acid sequence that does not contain lysine, which is present in the wild-type amino acid sequence. The variant linker sequence (variant first linker element) preferably does not contain lysine (K), more preferably is a sequence composed of amino acids other than lysine (K) and arginine (R), and preferably consists of one or more amino acids.

[0104] On the other hand, the protein of the second aspect in the first embodiment of the present invention is characterized in that, in the amino acid sequence of at least one B, C, and Z domains, the lysine at position 4 and the lysine at the C-terminus are missing or replaced. Therefore, by linking multiple such domains together, a variant linking position sequence (variant first linking element) of the third aspect is formed. Furthermore, when the domain located at the N-terminal side or the C-terminal side of the protein is such a domain, the terminal portion does not form a variant linking position sequence, but in the affinity separating agent obtained by immobilizing the affinity ligand onto the support described later, by immobilizing the N-terminal side or the C-terminal side of such a domain of the protein onto the support, the binding of the protein to the support and the binding of the immobilized domain to other domains can be stably maintained.

[0105] Here, in the first embodiment of the present invention, "variation" refers to changes resulting from partial substitution, insertion, deletion, and chemical modification of the wild-type amino acid sequence. "Varied linker sequence (varied first linker element)" refers to a sequence obtained by inserting a specific "variation" into the linker sequence (first linker element) of the wild-type amino acid sequence. Furthermore, "varied domain" refers to a domain obtained by inserting a specific "variation" into the wild-type amino acid sequence of each domain.

[0106] In the first embodiment of the present invention, at least one of the linker position sequences (first linker elements) contained in the proteins of the first and third methods may be a variant linker position sequence (variant first linker element). However, in the case of proteins of trimer or higher, it is preferable that two or more are variant linker position sequences (variant first linker elements). In the case of n-mer proteins, it is more preferable that n-1 are variant linker position sequences (variant first linker elements). In other words, it is more preferable that all linker position sequences (first linker elements) are variant linker position sequences (variant first linker elements).

[0107] Therefore, for the protein of the first embodiment of the present invention, it is preferable that lysine residues are missing or replaced in all the linker sequences (first linker elements).

[0108] In the affinity separation agent described later, in order to prevent the ligand immobilized on the support from being cleaved by the protease, it is preferable that the lysine at position 4 of the N-terminal domain or the C-terminal lysine at position C-terminus of the protein of the first embodiment of the present invention is missing or replaced.

[0109] Therefore, in the protein of the second aspect of the first embodiment of the present invention, at least one domain in which the lysine residue at position 4 and the lysine residue at the C-terminus are missing or replaced (a variant domain) is acceptable. However, it is preferable that the N-terminal domain or the C-terminal domain of the protein is a variant domain, and it is even more preferable that the fixed domain described later is a variant domain. In addition, in the case of trimer or higher proteins, it is preferable that the fixed domain and two or more domains connected thereto are variant domains. In the case of n-mer proteins, it is preferable that the fixed domain and n-2 or more domains connected thereto are variant domains, and most preferably all of the domains are variant domains.

[0110] The linker sequence (first linker element) consists of one amino acid residue at the C-terminus of the amino acid sequence of each domain and the sequence at positions 1 to 5. This sequence can be modified by substitution, insertion, deletion and chemical modification. It is preferably composed of more than one amino acid, and more preferably a sequence composed of amino acids other than lysine (K) and arginine (R).

[0111] According to the research of the inventors, it has been clarified that the site most easily cleaved by serine proteases such as trypsin and plasmin is the C-terminal side of a specific lysine (K) residue present in the wild-type amino acid sequence of the linker sequence (first linker element). It was found that if the linker sequence (variant first linker element) is composed of amino acids that do not contain the specific lysine (K) residue present in the linker sequence (first linker element), the cleavage of the protein by serine proteases can be significantly inhibited.

[0112] Specifically, a variant linking position sequence (variant first linking element) can be created by deleting and / or replacing K in the linking position sequence (first linking element) of the wild-type amino acid sequence. It is preferable to form a variant linking position sequence (variant first linking element) composed of amino acids other than K, and more preferably to form a variant linking element composed of amino acids other than K and R.

[0113] Furthermore, according to the research of the inventors, it has been found that K and R, which exist outside the linker sequence (first linker element) of the wild-type amino acid sequence, are not easily cleaved by serine proteases. Therefore, they can not only exist in each domain of the protein of the first embodiment of the present invention, but also, in order for the protein of the first embodiment of the present invention to have sufficient immunoglobulin binding activity, it is necessary for at least one domain to contain more than one lysine.

[0114] For lysine residues present outside the linker sequence (first linker element), it is preferable to retain lysine residues from the wild-type amino acid sequence, preferably two or more, more preferably three or more, and most preferably four lysine residues at the same position as the wild-type amino acid sequence. As for the retained K, it is most preferable to retain the lysine residue at position 35.

[0115] While the rationale for requiring lysine residues outside the linker sequence (first linker element) to achieve sufficient immunoglobulin binding activity is unclear, it is believed that lysine residues near the antibody recognition site of wild-type protein A interact electrostatically with the antibody. Therefore, lysine residues not present in the wild-type amino acid sequence can be included through substitutions, insertions, or other variations, without impairing the function of the antibody recognition site of wild-type protein A; however, it is preferable not to include lysine residues not present in the wild-type amino acid sequence.

[0116] The following is a specific example of a mutated connection position sequence (mutated first connection element).

[0117] When a variant linker sequence (variant first linker element) is created by deleting a portion of the amino acids in the linker sequence (first linker element), the variant linker sequence (variant first linker element) must consist of at least one amino acid. According to the research of the inventors, when all six amino acids contained in the linker sequence (first linker element) are deleted, the binding affinity to immunoglobulins is significantly reduced, and if this protein is used as an affinity ligand, the binding affinity to immunoglobulins is insufficient.

[0118] Generally, immunoglobulins are larger than their domains, and the steric hindrance between bound immunoglobulins limits the number of domains that can bind to each other. Therefore, it is speculated that this is because when each domain binds to an immunoglobulin, if there is no linker sequence (first linker element) and adjacent domains are too close together, there is insufficient space for immunoglobulin binding, resulting in a decrease in the number of immunoglobulins bound to each domain. If the modified linker sequence (first linker element) is preferably composed of one or more amino acids, more preferably two or more, the binding affinity to immunoglobulins tends to increase further, and is therefore preferred.

[0119] For the protein of the first embodiment of the present invention, the linker sequence (first linker element) is composed of a lysine (K) at the C-terminus of any domain of protein A and the sequences from position 1 to position 5 of the amino acid sequences of domains E, D, and A. Therefore, the linker sequence (first linker element) of the wild-type amino acid sequence contains one K. Therefore, it is preferable that the variant linker sequence (variant first linker element) is obtained by deleting the lysine (K) at the C-terminus of the linker domain.

[0120] For example, when a linker sequence (first linker element) formed by linking the A domains of two proteins A is made into a variant linker sequence (variant first linker element), preferred examples include a sequence consisting of five amino acids (alanine (A), aspartic acid (D), asparagine (N), asparagine (N), and phenylalanine (F)) with the C-terminal lysine (K) missing, or a sequence consisting of three amino acids (D, N, N) with the first A and the fifth F missing in addition to the C-terminal K deletion.

[0121] For the proteins of the second and third embodiments in the first embodiment of the present invention, the linker sequence (first linker element) is composed of a lysine (K) at the C-terminus of any domain of protein A and the sequences at positions 1 to 5 of the amino acid sequences of domains B, C, and Z. Therefore, the linker sequence (first linker element) of the wild-type amino acid sequence contains two Ks at the C-terminus and position 4. Therefore, it is preferable that the variant linker sequence (first linker element) omits the lysine (K) at the C-terminus and position 4 of the linked domain.

[0122] For example, when creating a variant linker sequence (variant first linker element) by linking the B domains of two proteins A, preferred examples include sequences consisting of alanine (A), aspartic acid (D), asparagine (N), and phenylalanine (F) with the C-terminal lysine (K) and the fourth lysine (K) missing; and sequences consisting of D and N with the C-terminal K and the fourth K missing, as well as the first V and the fifth F missing. Furthermore, when replacing one of the Ks in the linker sequence (first linker element) using the method described later, only the C-terminal K or only the fourth K can be missing.

[0123] Amino acid substitution refers to the mutation of removing an existing amino acid and adding another amino acid at the same position. There are no particular limitations on the other amino acids added; examples include naturally occurring protein-forming amino acids, non-forming protein amino acids, and non-natural amino acids. However, from a genetic engineering production perspective, naturally occurring amino acids are preferred.

[0124] When creating a variant linker sequence (variant first linker) by replacing amino acids in the linker sequence (first linker element), the variant linker sequence (variant first linker element) needs to be a sequence that does not contain lysine (K). If it is a sequence that further does not contain arginine (R), the resistance to proteases that recognize arginine is also improved, and therefore it is preferred.

[0125] Therefore, the amino acid introduced through substitution is not particularly limited as long as it is not lysine (K) and arginine (R), but hydrophilic amino acids such as asparagine (N), aspartic acid (D), glutamine (Q), glutamic acid (E), and histidine (H) are preferred; and any one of neutral amino acids such as cysteine ​​(C), glycine (G), methionine (M), serine (S), threonine (T), and tyrosine (Y) is preferred. Among these, any one of the hydrophilic amino acids is more preferred.

[0126] Furthermore, from the viewpoint of improving stability under alkaline conditions, aspartic acid (D), glutamine (Q), glutamic acid (E), and histidine (H) are preferred.

[0127] For the protein of the first embodiment of the present invention, the wild-type amino acid sequence contains a K in the linker position sequence (first linker element), and preferably the variant linker position sequence (variant first linker element) is formed by replacing the lysine (K) at the C-terminus of the linker domain.

[0128] For example, when a linker sequence (first linker element) formed by linking the A domains of two proteins A is made into a variant linker sequence (variant first linker element), it is preferable to replace the C-terminal lysine (K) with an amino acid other than lysine (K) and arginine (R).

[0129] Specifically, as preferred examples, sequences obtained by replacing the C-terminal lysine (K) with asparagine (N), aspartic acid (D), and histidine (H), and sequences obtained by replacing the C-terminal K, the first position A, and the fifth position F with asparagine (N), aspartic acid (D), and histidine (H), etc.

[0130] For the protein of the second manner in the first embodiment of the present invention, the wild-type amino acid sequence contains two Ks at the C-terminus and the fourth position in the linking position sequence (first linking element). Preferably, the variant linking position sequence (variant first linking element) is formed by replacing the lysine (K) at the C-terminus and the lysine (K) at the fourth position of the linking domain.

[0131] For example, when creating a variant linker sequence (variant first linker element) by linking the B domains of two proteins A, it is preferable to replace the C-terminal lysine (K) and the 4th lysine (K) with amino acids other than lysine (K) and arginine (R). Specifically, as preferred examples, sequences obtained by replacing the C-terminal lysine (K) and the 4th lysine (K) with asparagine (N), aspartic acid (D), and histidine (H), and sequences obtained by replacing the C-terminal K and the 4th K, as well as the 1st V and the 5th F with asparagine (N), aspartic acid (D), and histidine (H), are examples.

[0132] In addition, when a certain K in the connection position sequence (first connection element) is missing using the above method, only the K at the end of C can be replaced, or only the K at the 4th position can be replaced.

[0133] In the variant linker sequence (variant first linker element), additional amino acid sequences can be inserted on top of the five amino acids contained in the wild-type amino acid sequence.

[0134] In the protein of the first embodiment of the present invention, if a variant linker sequence (variant first linker element) is formed by deleting or replacing the C-terminal lysine (K) and / or the 4th position lysine (K), resistance to serine proteases can be improved. Furthermore, in the protein of the first embodiment of the present invention, if a variant linker sequence (variant first linker element) consisting of four amino acids is formed by deleting the terminal K and / or the 4th position K, resistance to serine proteases such as trypsin and plasmin can be further improved, and is therefore preferred.

[0135] Furthermore, although the reason is not yet certain, if a variant linker sequence (variant first linker element) is created by deleting several amino acids other than K in the linker sequence (first linker element) of the wild-type amino acid sequence, preferably consisting of 3 amino acids, more preferably 2 amino acids, then the serine protease resistance tends to be further improved, and is therefore preferred. It is speculated that this is because shortening the linker sequence (first linker element) inhibits physical contact with the protease.

[0136] In addition to being resistant to serine proteases, the protein of the first embodiment of the present invention may also be resistant to other proteases. For example, when the variant linker sequence (variant first linker element) is composed of amino acids other than hydrophobic amino acids, it is resistant to thermophilic proteases and is therefore preferred.

[0137] There are no particular restrictions on the amino acid sequence introduced outside the mutant linker sequence (mutant first linker element), as long as the affinity for the Fab region of immunoglobulins is reduced to the same or greater than that of proteins with wild-type amino acid sequences, and the proteins have an affinity for immunoglobulins.

[0138] The amino acid sequence of the portion other than the mutated linker position sequence (mutated first linker element) has preferably 85% or more, more preferably 90% or more sequence homology with the wild-type amino acid sequence of any one of the E, D, A, B, C and Z domains of protein A. As a result, the protein of the first embodiment of the present invention can have the ability to bind to the Fc region.

[0139] The domains of the protein obtained by introducing mutations show preferably 85% or more, more preferably 90% or more, amino acid sequence homology with any one of the wild-type amino acid sequences of the E, D, A, B, C and Z domains of protein A.

[0140] As a specific example of the aforementioned proteins, proteins consisting of the amino acid sequences recorded in sequence numbers 48 to 57 can be cited.

[0141] The protein of the first embodiment of the present invention is characterized by its resistance to proteases, particularly serine proteases. Examples of serine proteases include trypsin and plasmin, which are typically proteases generated and accumulated in microorganisms such as Escherichia coli and cultured cells such as CHO cells.

[0142] For example, when producing the protein according to the first embodiment of the present invention, typically, as described below, a transformant using a microorganism such as Escherichia coli as a host is used to accumulate the protein within or outside the cells of the transformant for recovery. The accumulation and recovery of the target protein is sometimes affected by proteases produced by the host microorganism.

[0143] Furthermore, when the protein of the first embodiment of the present invention is used as an affinity separating agent to purify immunoglobulins, it is sometimes affected by the protease contained in the culture supernatant of CHO cells or the like that produce immunoglobulins via the affinity separating agent.

[0144] According to the research of the inventors, for multimeric proteins with wild-type amino acid sequences, the protein is cleaved by proteases during the accumulation and recovery of the multimeric protein, and the target multimeric protein cannot be obtained.

[0145] Furthermore, it is known that when multimeric proteins with wild-type amino acid sequences are used as ligands to prepare affinity separation agents for the purification of immunoglobulins, the multimeric proteins acting as ligands are cleaved by proteases, thus failing to fully exert their immunoglobulin binding capacity. This is the first time it has been discovered that this is due to the influence of serine proteases produced by microorganisms such as E. coli and cultured cells such as CHO cells, where the amino acid sequences of each domain are cleaved at specific points.

[0146] Based on these research results, the protein of the first embodiment of the present invention, obtained by adding specific changes to the linker position sequence (first linker element), is not cleaved by serine proteases during culture and in purification and concentration processes after recovery, and can be accumulated and recovered.

[0147] Furthermore, when the multimeric protein of the first embodiment of the present invention is used as a ligand to prepare an affinity separating agent, it is not affected by serine proteases and can exert sufficient immunoglobulin binding to purify the target immunoglobulin.

[0148] In order to significantly improve the effect of the ligand functioning stably without being affected by proteases when used in the purification process of immunoglobulins, the protein of the first embodiment of the present invention preferably contains one or more non-fixed structural domains that do not bind to the carrier, more preferably two or more, and in the case of an n-mer protein, preferably n-1 or more are non-fixed structural domains, and most preferably all n are non-fixed structural domains.

[0149] In order to make the protein of the first embodiment of the present invention include the non-fixed structural domains described in detail below, it can be made by introducing amino acid residues useful for fixation into the amino acid sequence of at least one or more structural domains by substitution, insertion, or deletion of amino acids useful for fixation, or by introducing fixation tags.

[0150] By including a non-fixed domain in the protein of the first embodiment of the present invention, the orientation can be adjusted and fixed to the support, thereby better exerting immunoglobulin binding.

[0151] Furthermore, even when the affinity separating agent of the first embodiment of the present invention is used repeatedly, the immunoglobulin binding of the ligand will not decrease, and immunoglobulins can be effectively purified.

[0152] Therefore, the protein of the first embodiment of the present invention has sufficiently high binding activity to the Fc region of immunoglobulin, making it suitable not only as an affinity ligand but also as a stable ligand that can be repeatedly used, which is advantageous in industry.

[0153] Here, in this specification, the presence of serine protease resistance is confirmed by the following method: A solution obtained by adding 2 μL of a 1–10 mg / mL serine protease solution to 10 μL of a 2 mg / mL ligand solution and heating at 37°C for 15 hours is preferred if as many ligands as possible remain uncleaved. The concentration of the serine protease solution can be appropriately set according to the type of protease; for example, trypsin is preferably 1 mg / mL, and plasmin is preferably 10 mg / mL.

[0154] As an indicator of serine protease resistance, it retains 80% or more, preferably 85% or more, and more preferably 90% or more of the uncleaved ligands. It should be noted that whether or not ligands are cleaved is confirmed by a commonly used electrophoresis method (SDS-PAGE).

[0155] [Second Embodiment of the Invention]

[0156] <Protein>

[0157] The protein of the second embodiment of the present invention is characterized in that it has two or more domains derived from any one of the A, B, C and Z domains of protein A described in sequence numbers 3 to 6, and at least one of the linking elements (second linking elements) connecting each domain is a variant linking element (variant second linking element) composed of one or more amino acids and composed of amino acids other than hydrophobic amino acids.

[0158] Protein A is a protein composed of five immunoglobulin-binding domains linked together. Many microorganisms express protein A; for example, Staphylococcus aureus expresses protein A.

[0159] The A, B, C and Z domains of protein A described in sequences 3 to 6 are immunoglobulin-binding proteins capable of binding to regions other than the complementarity-determining region (CDR) of immunoglobulins. Each domain can bind to various regions of immunoglobulins, such as the Fc region, Fab region and especially the Fv region of the Fab region.

[0160] like Figure 1 As shown in the sequence comparison table, the A, B, C, and Z domains derived from protein A have highly similar amino acid sequences, exhibiting over 80% amino acid sequence homology. It should be noted that hyphens (-) indicate amino acid residues identical to those in the C domain.

[0161] "A domain derived from any one of the A, B, C, and Z domains of protein A" refers to a domain having the following amino acid sequence derived from the amino acid sequence of each domain in the wild type. As long as it can encode a protein capable of binding to the Fc region, variations other than the variant linker element (variant second linker element) described later can be introduced into the amino acid sequence of each domain in the wild type.

[0162] In other words, the amino acid sequence of any one of the A, B, C, and Z domains of protein A is the amino acid sequence before the introduction of the mutation (wild-type amino acid sequence), while the amino acid sequence "derived from any one of the A, B, C, and Z domains of protein A" refers to the wild-type amino acid sequence of the A, B, C, and Z domains themselves, or the amino acid sequence that contains the mutant linker element (mutant second linker element) described later and / or has been altered by partial substitution, insertion, deletion, and chemical modification of amino acids.

[0163] Here, the Z domain of protein A is obtained by introducing variations such as A1V and G29A into the B domain. It is a domain that does not exist in the natural protein A. However, in this specification, the amino acid sequence represented by sequence number 6 is referred to as the wild-type amino acid sequence of the Z domain.

[0164] The protein of the second embodiment of the present invention is a multimeric protein (multi-domain protein) having two or more of the aforementioned "domains derived from any one of the A, B, C, and Z domains of protein A" as single domains. Preferably, there are three or more, more preferably four or more, more preferably ten or less, more preferably eight or less, and even more preferably six or less.

[0165] These multimeric proteins can be linkers of a single immunoglobulin-binding domain, i.e., homodimers, homotrimers, or other homopolymers, or linkers of different types of immunoglobulin-binding domains, i.e., heterodimers, heterotrimers, or other heteropolymers. Preferably, the protein of the second embodiment of the present invention comprises homopolymers whose domains are all derived from any one of the amino acid sequences of the A, B, C, and Z domains of protein A as described in sequences 3 to 6.

[0166] For the protein of the second embodiment of the present invention, the C-terminus and N-terminus of two or more structural domains are connected to form a linker element (second linker element). Here, in the second embodiment of the present invention, "linker element (second linker element)" refers to the sequence of the 1st to 5th positions in the amino acid sequence of each structural domain.

[0167] In other words, in a domain that connects to other domains at its N-terminus, the sequence from position 1 to position 5 of the amino acid sequence of that domain becomes a linker element (second linker element). Therefore, there are n-1 linker elements (second linker elements) in an n-mer protein.

[0168] The sequences shown below are the amino acid sequences of the A, B, C, and Z domains of protein A, from position 1 to position 5. Each sequence consists of 5 amino acids, including alanine (A) and / or phenylalanine (F), which are hydrophobic amino acids.

[0169]

[0170] The protein of the second embodiment of the present invention is characterized in that at least one linker element (second linker element) is a variant linker element (variant second linker element) altered by partial substitution, insertion, deletion, and chemical modification of amino acids. The variant linker element (variant second linker element) is a sequence consisting of one or more amino acids, and consisting of amino acids other than hydrophobic amino acids.

[0171] In the second embodiment of the present invention, at least one of the linking elements (second linking elements) contained in the protein may be a variant linking element (variant second linking element), but in the case of a trimer or more proteins, it is preferable that two or more are variant linking elements (variant second linking elements), and in the case of an n-mer protein, it is more preferable that n-1 are variant linking elements (variant second linking elements). In other words, it is more preferable that all the linking elements (second linking elements) are variant linking elements (variant second linking elements).

[0172] Linking elements (second linking elements) are composed of the sequence from position 1 to position 5 of the amino acid sequence of each domain. However, sequences that are altered by substitution, insertion, deletion, or chemical modification and are composed of more than one amino acid and are composed of amino acids other than hydrophobic amino acids are variant linking elements (variant second linking elements).

[0173] According to the research of the inventors, it has been found that the site most easily cleaved by thermophilic proteases is the N-terminal side of the hydrophobic amino acid in the linker element (second linker element). Therefore, if the linker element (second linker element) is made into a variant linker element (variant second linker element) composed of amino acids other than hydrophobic amino acids, the cleavage of proteins by thermophilic proteases can be significantly inhibited.

[0174] Specifically, it is preferable to create a variant linker (variant second linker) by deleting and / or replacing A and F in the linker element (second linker element) of the wild-type amino acid sequence.

[0175] Here, "hydrophobic amino acids" in this specification refer to alanine (A), isoleucine (I), leucine (L), phenylalanine (F), proline (P), tryptophan (W), and valine (V). "Amino acids other than hydrophobic amino acids" refer to hydrophilic amino acids such as arginine (R), asparagine (N), aspartic acid (D), glutamine (Q), glutamic acid (E), histidine (H), and lysine (K); naturally occurring neutral amino acids such as cysteine ​​(C), glycine (G), methionine (M), serine (S), threonine (T), and tyrosine (Y); and non-natural neutral amino acids such as acetyllysine, azide-Z-lysine, glutamic acid 5-methyl ester, and aspartic acid 5-methyl ester.

[0176] The following describes a specific example of a variant connecting element (variant second connecting element).

[0177] When a variant linker (variant second linker) is created by deleting a portion of the amino acids in the linker element (second linker), the variant linker element (variant second linker) must consist of at least one amino acid. According to the research of the inventors, when all five amino acids contained in the linker element (second linker) are deleted, the binding affinity to immunoglobulins is significantly reduced, and if this protein is used as an affinity ligand, the binding affinity to immunoglobulins is insufficient. This difference is particularly pronounced depending on the purification conditions used as the affinity separating agent.

[0178] Generally, immunoglobulins are larger than their domains, and the steric hindrance between bound immunoglobulins limits the number of domains that can bind. Therefore, it is speculated that this is because, when each domain binds to an immunoglobulin, if there is no linker element (second linker element) and adjacent domains are too close together, there is insufficient space for immunoglobulin binding, resulting in a reduced number of immunoglobulins bound per domain. Considering the excellent binding affinity to immunoglobulins under various purification conditions, the variant linker element (variant second linker element) must consist of one or more amino acids; if it consists of two or more amino acids, the binding affinity to the target immunoglobulin tends to increase, and therefore this is preferred.

[0179] When the variant linker element (variant second linker element) is included in any of the A, B, and C domains derived from protein A, it is preferred that the variant linker element (variant second linker element) is composed of the sequence of the first to fifth positions of the amino acid sequence of the A, B, or C domain, and that the alanine at the first position and / or the phenylalanine at the fifth position are deleted.

[0180] For example, preferred examples include sequences consisting of aspartic acid (D), asparagine (N), and lysine (K) with the amino acids alanine (A) at position 1 and phenylalanine (F) at position 5 missing; and sequences consisting of D and N with the amino acids alanine (A) at position 1 and F at position 5 missing, and K at position 4 missing. Furthermore, when replacing the hydrophobic amino acid contained in the linker element (the second linker element) using the method described later, either only A at position 1 or only F at position 5 can be missing.

[0181] When the variant linker (variant second linker) is included in a domain derived from the Z domain of protein A, the variant linker (variant second linker) preferably consists of the sequence of the first to fifth amino acid positions in the Z domain and omits the valine at the first position and / or the phenylalanine at the fifth position.

[0182] For example, preferred examples include sequences consisting of aspartic acid (D), asparagine (N), and lysine (K) with the amino acids valine (V) at position 1 and phenylalanine (F) at position 5 missing; and sequences consisting of D and N with the amino acids valine (V) at position 1 and F at position 5 missing, and K at position 4 missing. Furthermore, when replacing a portion of the hydrophobic amino acid in the linker element (second linker element) using the method described later, only V at position 1 or only F at position 5 may be missing.

[0183] Amino acid substitution refers to the mutation of removing an existing amino acid and adding another amino acid at the same position. There are no particular limitations on the other amino acids added; examples include naturally occurring protein-forming amino acids, non-protein-forming amino acids, and non-natural amino acids. However, from a genetic engineering production perspective, naturally occurring amino acids are preferred.

[0184] When a variant linker (variant second linker) is created by replacing an amino acid in the linker element (second linker element), the variant linker element (variant second linker element) must have a sequence that does not contain hydrophobic amino acids. Therefore, the amino acid introduced through substitution is not particularly limited as long as it is an amino acid other than a hydrophobic amino acid, and is preferably a hydrophilic amino acid such as arginine (R), asparagine (N), aspartic acid (D), glutamine (Q), glutamic acid (E), histidine (H), and lysine (K); or any one of neutral amino acids such as cysteine ​​(C), glycine (G), methionine (M), serine (S), threonine (T), and tyrosine (Y). More preferably, it is any one of the hydrophilic amino acids.

[0185] Furthermore, from the viewpoint of improving stability under alkaline conditions, arginine (R), aspartic acid (D), glutamine (Q), glutamic acid (E), histidine (H), and lysine (K) are preferred.

[0186] In addition, from the viewpoint of improving tolerance to other proteases, such as serine proteases described later, aspartic acid (D), glutamine (Q), glutamic acid (E), and histidine (H) are preferred.

[0187] When the variant linker (variant second linker) is included in any one of the A, B, and C domains derived from protein A, it is preferable that the variant linker (variant second linker) is composed of the sequence of the first to fifth positions of the amino acid sequence of the A, B, or C domain, and that the alanine at the first position and / or the phenylalanine at the fifth position are replaced by an amino acid other than a hydrophobic amino acid.

[0188] For example, as preferred examples, sequences obtained by replacing the two amino acids at position 1 (alanine (A)) and position 5 (phenylalanine (F)) with amino acids other than hydrophobic amino acids can be given, as can sequences obtained by replacing the first position (A), the fifth position (F), and the fourth position (K) with amino acids other than K that are hydrophobic. Furthermore, when a portion of the hydrophobic amino acids contained in the linker element (the second linker element) is omitted using the method described above, only the first position (A) or only the fifth position (F) can be replaced.

[0189] When a variant linker (variant second linker) is included in a domain derived from the Z domain of protein A, it is preferable that the variant linker (variant second linker) is composed of the sequence of the first to fifth amino acid positions in the Z domain and that the valine at the first position and / or the phenylalanine at the fifth position are replaced by an amino acid other than a hydrophobic amino acid.

[0190] For example, as preferred examples, sequences obtained by replacing valine (V) at position 1 and phenylalanine (F) at position 5 with amino acids other than hydrophobic amino acids, and sequences obtained by replacing V at position 1, F at position 5, and K at position 4 with amino acids other than K that are hydrophobic, are also examples. Furthermore, when a portion of the hydrophobic amino acids contained in the linker element (the second linker element) is omitted using the method described above, only V at position 1 or only F at position 5 can be replaced.

[0191] In the variant linker element (variant second linker element), in addition to the five amino acids contained in the wild-type amino acid sequence, additional amino acid sequences may be inserted. For example, a variant linker element (variant second linker element) consisting of six or more amino acids may be made by further inserting amino acids other than hydrophobic amino acids into the sequence obtained by replacing a portion of the hydrophobic amino acids contained in the linker element (second linker element) using the method described above.

[0192] Alternatively, some or all of the amino acids can be further deleted from the sequence obtained by deleting a portion of the hydrophobic amino acids contained in the linker element (second linker element) using the method described above, and any variant linker element (variant second linker element) composed of amino acids other than hydrophobic amino acids can be inserted. In addition, chemical modifications such as acetylation of lysine residues and hydroxylation of proline residues can be performed without affecting the binding affinity to antibodies.

[0193] If the three-dimensional structure of the obtained protein changes significantly, it may alter the affinity and binding ability of the target immunoglobulin. Therefore, it is preferable not to insert additional amino acid sequences into the linker element (second linker element) of the wild-type amino acid sequence.

[0194] The amino acid constituting the variant linking element (variant second linking element) that has been mutated by substitution, insertion, or other means is not particularly limited as long as it is an amino acid other than a hydrophobic amino acid. Preferred amino acids are hydrophilic amino acids such as arginine (R), asparagine (N), aspartic acid (D), glutamine (Q), glutamic acid (E), histidine (H), and lysine (K), and more preferably asparagine (N), aspartic acid (D), and histidine (H).

[0195] There are no particular restrictions on the amino acid sequence changes introduced outside of the variant linker element (variant second linker element), as long as they have the same or greater affinity and binding capacity for immunoglobulins compared to proteins with wild-type amino acid sequences.

[0196] The amino acid sequence of the portion other than the variant linker element (variant second linker element) has preferably 85% or more, more preferably 90% or more sequence homology with the wild-type amino acid sequence of any one of the A, B, C and Z domains of protein A. As a result, the protein of the second embodiment of the present invention can have the ability to bind to the Fc region.

[0197] The domains of the protein obtained by introducing the mutation show preferably more than 85%, more preferably more than 90% sequence homology with any one of the wild-type amino acid sequences of the A, B, C and Z domains of protein A.

[0198] Besides affinity and binding ability to immunoglobulins, other useful ligand properties include alkali resistance and weak acid solubility. To improve these properties, modifying the amino acid sequence other than the variant linker element (variant second linker element) is a preferred approach in this invention.

[0199] Alkali resistance refers to the ability to maintain high affinity and binding capacity with immunoglobulins when immersed in alkaline solutions such as 0.1N NaOH or 0.5N NaOH. Therefore, when using alkali-resistant proteins as affinity ligands to prepare separating agents, high separation and purification capabilities can be maintained even after repeated washing with alkaline solutions. It should be noted that the C and Z domains are known to inherently possess high alkali resistance and are preferred sequences in terms of alkali resistance.

[0200] Furthermore, weakly acidic solubility refers to the ability of immunoglobulins, which typically dissolve under acidic conditions, to dissolve under milder conditions at a higher pH when purified by adsorption with a separating agent immobilized with affinity ligands. This allows for the inhibition of denaturation, such as protein aggregation, during dissolution. The variations in weakly acidic solubility described in Patent Document 5, International Publication No. 2010 / 118699, etc., are all incorporated into this invention, making it a preferred embodiment.

[0201] As a specific example of the aforementioned proteins, proteins consisting of the amino acid sequences recorded in sequence numbers 105-115 can be cited.

[0202] The protein of the second embodiment of the present invention is characterized by its resistance to proteases, particularly thermophilic bacterial proteases. When preparing the protein of the second embodiment of the present invention, as described below, a transformant with a microorganism as the host is typically used to accumulate the protein within or outside the cells of the transformant for recovery. During the accumulation and recovery of the target protein, it is sometimes affected by proteases produced by the host microorganism.

[0203] According to the research of the inventors, when accumulating and recovering multimeric proteins with wild-type amino acid sequences using the above-described method, the sequences are cleaved by proteases during the culturing process and in the purification and concentration processes after recovery, resulting in the failure to obtain the target multimeric protein. This is the first time it has been discovered that this is due to the influence of thermophilic proteases produced by bacteria such as Bacillus, which cleave specific portions of the amino acid sequences in each domain.

[0204] In light of these findings, the protein of the second embodiment of the present invention, with specific modifications incorporated into the linker element (second linker element), is not cleaved by thermophilic proteases during culture and in purification and concentration processes after recovery, enabling accumulation and recovery. Therefore, the protein of the second embodiment of the present invention not only possesses sufficiently high binding activity to the Fc region of immunoglobulins, making it suitable as an affinity ligand, but also can be produced with high efficiency, which is industrially advantageous.

[0205] Here, in this specification, the presence of thermophilic protease resistance is confirmed by the following method: A solution obtained by adding 2 μL of a 1 mg / mL thermophilic protease solution to 10 μL of a 2 mg / mL ligand solution, and then heating at 37°C for 15 minutes, is preferred if as many ligands as possible remain uncleaved. For example, it is preferable to retain 80% or more, more preferably 85% or more, and more preferably 90% or more of the uncleaved ligands. It should be noted that whether ligands are cleaved is confirmed using a commonly used electrophoresis method (SDS-PAGE).

[0206] The protein of the second embodiment of the present invention is resistant to thermophilic proteases by having a variant linker element (variant second linker element), but preferably also has an amino acid sequence that is resistant to other proteases. Examples of other proteases include serine proteases such as trypsin and plasmin.

[0207] When using the protein of the second embodiment of the present invention as an affinity separating agent for the ligand to purify IgG antibodies produced from recombinant cultured cells, the ligand is cleaved by serine proteases produced by the cultured cells, which may prevent it from exerting sufficient immunoglobulin binding. Therefore, it is preferable that the protein of the second embodiment of the present invention has an amino acid sequence that is also resistant to serine proteases.

[0208] For the reasons stated above, it is preferable that at least one domain's C-terminal lysine residue is missing or substituted. According to the research of the inventors, it is known that the site most easily cleaved by serine proteases such as trypsin and plasmin is the C-terminal side of the C-terminal lysine (K) present in the wild-type amino acid sequence of each domain. Therefore, it has been found that if the amino acid sequence consists of a C-terminal lysine (K) residue that is bound by a missing or substituted linker element (second linker element) of each domain, the cleavage of the protein by serine proteases can be significantly inhibited.

[0209] More preferably, in the domain on the C-terminal side where the aforementioned variant linker element (variant second linker element) is bound, the C-terminal lysine in the amino acid sequence of that domain is missing or replaced.

[0210] Further preferred variant linker element (variant second linker element) also does not contain lysine. As mentioned above, for the wild-type amino acid sequence of the B, C, and Z domains, lysine is contained at position 4 in the linker element (second linker element).

[0211] According to the research of the inventors, lysine in the linker element (second linker element) is more easily cleaved by serine proteases. Therefore, it is preferable that the modified linker element (modified second linker element) has an amino acid sequence that does not contain lysine (K) through deletion or substitution. It is further preferred that the modified linker element (modified second linker element) does not contain arginine (R).

[0212] The following describes specific examples of variant linkers with serine protease resistance.

[0213] Examples include the deletion of a portion of the C-terminal lysine (K) of each domain and / or the amino acid of the linker element (second linker element). When the linker element (second linker element) is composed of the sequence from position 1 to position 5 of the amino acid sequence of domain A, only the C-terminal K of each domain needs to be deleted.

[0214] On the other hand, when the linking element (second linking element) is composed of the sequence of the amino acid sequence of the B, C and Z domains from position 1 to position 5, it includes two Ks at the C-terminus and position 4. Preferably, at least the K at the C-terminus is missing, and more preferably, the K in the linking element (second linking element) is also missing.

[0215] For example, when the linker element (second linker element) formed by connecting the B domains of two proteins A is a variant linker element (variant second linker element) created by deleting a hydrophobic amino acid, as preferred examples include a sequence consisting of three amino acids: aspartic acid (D), asparagine (N), and lysine (K) with the C-terminal lysine (K) further deleted, and a sequence consisting of two amino acids: aspartic acid (D) and asparagine (N) with the C-terminal lysine (K) and the lysine (K) at position 4 further deleted.

[0216] In addition, when replacing either the K at the end of C or the K contained in the connecting element (the second connecting element) using the method described later, it is possible to omit only the K at the end of C or only the K in the fourth position.

[0217] When replacing a portion of the amino acid at the C-terminal lysine (K) of each domain and / or the linker element (second linker element), examples include removing the original amino acid and adding another amino acid at the same position. The added amino acid is not particularly limited; examples include native protein-forming amino acids, non-protein-forming amino acids, and non-native amino acids. From the viewpoint of genetic engineering production, native amino acids are preferred.

[0218] When a variant linker (variant second linker) is created by replacing an amino acid in the linker element (second linker element), it must be composed of amino acids other than hydrophobic amino acids. Preferably, the variant linker (variant second linker element) does not contain a sequence such as lysine (K). If it is a sequence that also does not contain arginine (R), its resistance to proteases that recognize arginine is also improved, and therefore it is preferred.

[0219] Therefore, the amino acid introduced through substitution is not particularly limited as long as it is not a hydrophobic amino acid, lysine (K), or arginine (R). Hydrophilic amino acids such as asparagine (N), aspartic acid (D), glutamine (Q), glutamic acid (E), and histidine (H) are preferred; as are any one of neutral amino acids such as cysteine ​​(C), glycine (G), methionine (M), serine (S), threonine (T), and tyrosine (Y). Among these, any one of the aforementioned hydrophilic amino acids is more preferred. Furthermore, from the viewpoint of improving stability under alkaline conditions, aspartic acid (D), glutamine (Q), glutamic acid (E), and histidine (H) are preferred.

[0220] When the linker element (second linker element) is composed of the sequence from position 1 to position 5 of the amino acid sequence of the A domain, only the K at the C-terminus of each domain may be replaced. On the other hand, when the linker element (second linker element) is composed of the sequence from position 1 to position 5 of the amino acid sequences of the B, C, and Z domains, including the two Ks at the C-terminus and position 4, it is preferable to replace at least the K at the C-terminus, and more preferably to replace all the Ks in the linker element (second linker element).

[0221] For example, when the linker element (second linker element) formed by connecting the B domains of two proteins A is a variant linker element (variant second linker element) created by replacing hydrophobic amino acids, it is preferable to further replace the C-terminal lysine (K) with an amino acid other than hydrophobic amino acid, lysine (K), and arginine (R). More preferably, it is preferable to further replace the C-terminal lysine (K) and the 4th position lysine (K) with an amino acid other than hydrophobic amino acid, lysine (K), and arginine (R). Specifically, as a preferred example, sequences obtained by replacing the V at position 1 and the F at position 5, as well as the K at position 4 and the K at position 1 with asparagine (N), aspartic acid (D), and histidine (H) are given.

[0222] In addition, when the K at the end of C and one of the Ks contained in the connecting element (the second connecting element) are missing using the above method, you can replace only the K at the end of C or only the K in the fourth position.

[0223] By incorporating the aforementioned variations, the protein of the second embodiment of the present invention is resistant to serine proteases. Examples of serine proteases include trypsin and plasmin, which are typically proteases generated and accumulated by microorganisms such as Escherichia coli and cultured cells such as CHO cells.

[0224] When the protein of the second embodiment of the present invention is used as an affinity separator to purify immunoglobulins, it is sometimes affected by the protease contained in the culture supernatant of antibody-producing CHO cells or the like, which are separated by the affinity separator.

[0225] According to the research of the inventors, it was discovered for the first time that the amino acid sequences of each domain are cleaved at specific locations due to the influence of serine proteases. In view of these findings, when the protein of the second embodiment of the present invention, with its amino acid sequence modified to be resistant to serine proteases, is used as a ligand to prepare an affinity separating agent, it will not be affected by serine proteases, and can exert sufficient immunoglobulin binding to purify the target immunoglobulin.

[0226] Furthermore, even when the affinity separating agent of the second embodiment of the present invention is used repeatedly, the immunoglobulin binding of the ligand does not decrease, and immunoglobulins can be effectively purified.

[0227] Therefore, the protein of the second embodiment of the present invention not only has sufficiently high binding activity to the Fc region of immunoglobulins, making it suitable for use as an affinity ligand, but is also stable as a ligand and can be reused repeatedly, which is advantageous in industry.

[0228] Here, in this specification, the presence of serine protease resistance is confirmed by the following method: A solution obtained by adding 2 μL of a 1–10 mg / mL serine protease solution to 10 μL of a 2 mg / mL ligand solution, and then heating at 37°C for 15 hours, should be heated. The more ligands that remain uncleaved, the better. The concentration of the serine protease solution can be appropriately set according to the type of protease; for example, trypsin is preferably 1 mg / mL, and plasmin is preferably 10 mg / mL.

[0229] As an indicator of serine protease resistance, it is preferable to retain 80% or more, more preferably 85% or more, and more preferably 90% or more of the uncleaved ligands. It should be noted that whether or not ligands are cleaved is confirmed by a commonly used electrophoresis method (SDS-PAGE).

[0230] <Methods for manufacturing proteins>

[0231] The following describes the manufacturing methods of the proteins (proteins of the present invention) according to the first and second embodiments of the present invention.

[0232] The protein of the present invention is produced by creating DNA having the base sequence encoding the aforementioned protein and translating the DNA. Specifically, it can be produced using a transformant obtained by transforming a microorganism as a host using a vector containing the DNA, or using a cell-free protein synthesis system employing the DNA.

[0233] DNA having the base sequence encoding the protein of the present invention can be obtained using commonly used and known methods such as polymerase chain reaction (hereinafter referred to as PCR). Alternatively, it can be synthesized using known chemical synthesis methods, or obtained from a genomic DNA library. The codons constituting the base sequence of this DNA can be replaced with degenerate codons, and need not be identical to the original base sequence, as long as they encode the same amino acids during translation.

[0234] As a method for specifically mutating the DNA that encodes the protein of the present invention at the insertion site, gene recombination technology, PCR, etc. can be used.

[0235] That is, the introduction of mutations using gene recombination technology can be carried out, for example, by using the cassette mutation method. In this method, if there are appropriate restriction endonuclease recognition sequences on both sides of the target site where the mutation is to be introduced in the gene encoding the protein of the present invention, the restriction endonuclease recognition sequence portions are cut with the restriction endonuclease, and after removing the region containing the site where the mutation is to be introduced, a DNA fragment in which the mutation has been introduced only at the target site through chemical synthesis or the like is inserted.

[0236] In addition, the introduction of site-specific variants using PCR can be performed, for example, using a double-primer method, that is, using a double-stranded plasmid encoding a protein as a template and performing PCR with two synthetic oligonucleotide primers that are complementary to the + and - strands and contain the variants.

[0237] Alternatively, DNA encoding the monomeric protein (one domain) of the present invention can be tandemly linked in the desired number to create DNA encoding a multimeric protein. For example, the ligation method for the DNA encoding the multimeric protein can be to introduce appropriate restriction endonuclease cleavage sites into the DNA sequence and ligate the restriction endonuclease-fragmented double-stranded DNA using DNA ligase. The restriction endonuclease cleavage sites can be of one type or multiple different types.

[0238] Methods for creating DNA encoding multimeric proteins are not limited to these linking methods. For example, the above-described mutation introduction method can also be applied to DNA encoding protein A (e.g., International Publication No. 2006 / 004067).

[0239] Furthermore, in DNA encoding multimeric proteins, when the base sequences encoding each monomeric protein are identical, homologous recombination in the host may occur. Therefore, it is preferable that the sequence homology between the base sequences of the DNA encoding the linked monomeric proteins is 90% or less, more preferably 85% or less.

[0240] The vector comprises: DNA containing a base sequence encoding the aforementioned protein or a portion thereof; and a promoter that is functionally linked to the base sequence and can function in the host. It is typically obtained by ligating or inserting DNA containing a gene encoding the aforementioned protein into a suitable vector.

[0241] There are no particular restrictions on the vectors used for gene insertion, as long as they can replicate autonomously within the host. Plasmid DNA and bacteriophage DNA can be used as vectors. For example, when using E. coli as a host, vectors such as the pQE series (manufactured by QIAGEN), pET series (manufactured by Merck), and pGEX series (manufactured by GE HealthCare Japan) can be used.

[0242] When using Bacillus brevis bacteria as a vector for transformation, examples include pUB110 or pHY500 (Japanese Patent Application Publication No. 2-31682), pNY700 (Japanese Patent Application Publication No. 4-278091), pNU211R2L5 (Japanese Patent Application Publication No. 7-170984), pHT210 (Japanese Patent Application Publication No. 6-133782), which are known as vectors for Bacillus subtilis, or pNCMO2 (Japanese Patent Application Publication No. 2002-238569), which is used as a shuttle vector for Escherichia coli and Bacillus brevis bacteria.

[0243] Transformants can be obtained by introducing a vector containing DNA encoding the protein of the present invention into a host cell. Methods for transforming the vector introduced into the host include, but are not limited to, methods using calcium ions, electroporation, protoplast methods, lithium acetate methods, Agrobacterium infection methods, gene gun methods, or polyethylene glycol methods.

[0244] In addition, as a method for maintaining a vector within a host, examples include methods that maintain the vector independently of the genome (chromosome) within the cell through autonomous replication; and methods that recombine a created gene into the genome (chromosome) and rely on genome replication for maintenance.

[0245] The host cell is not particularly limited, but from the perspective of inexpensive mass production, bacteria (eubacteria) such as *Escherichia coli*, *Bacillus subtilis*, *Bacillus brevis*, *Staphylococcus*, *Streptococcus*, *Streptomyces*, and *Corynebacterium* are preferred. More preferably, Gram-positive bacteria such as *Bacillus subtilis*, *Bacillus brevis*, *Staphylococcus*, *Streptococcus*, *Streptomyces*, and *Corynebacterium* are preferred. Even more preferred are *Bacillus brevis* bacteria known in examples of mass production of protein A (International Publication No. 2006 / 004067).

[0246] As bacteria of the genus *Brevibacillus*, there is no limitation, but examples include *Brevibacillus spp.*, *Brevibacillus borstelensis*, *Brevibacillus brevis*, *Brevibacillus centrosporus*, *Brevibacillus choshinensis*, *Brevibacillus formosus*, *Brevibacillus invocatus*, *Brevibacillus laterosporus*, *Brevibacillus limnophilus*, *Brevibacillus parabrevis*, *Brevibacillus reuszeri*, and *Brevibacillus thermophilus*.

[0247] Preferred examples include Bacillus brevis strain 47 (JCM6285), Bacillus brevis strain 47K (FERM BP-2308), Bacillus brevis strain 47-5Q (JCM8970), Bacillus brevis HPD31 (FERM BP-1087), and Bacillus brevis HPD31-OK (FERM BP-4573).

[0248] For purposes such as increasing production, mutant strains (or induced strains) of the aforementioned *Bacillus* genus, such as protease-deficient strains, high-expression strains, or strains lacking spore-forming ability, can be used. Specifically, protease mutant strains derived from *Bacillus chinensis* HPD31, namely *Bacillus chinensis* HPD31-OK (Japanese Patent Application Publication No. 6-296485), and *Bacillus chinensis* HPD31-SP3 (International Publication No. 2005 / 045005), which lacks spore-forming ability, can be used.

[0249] The protein of the present invention can be manufactured using a transformant or a cell-free protein synthesis system using the aforementioned DNA.

[0250] When producing proteins using transformants, the proteins can be accumulated within the cells of the transformant (including the periplasmic region) or recovered from the culture solution (extracellularly). Intracellular accumulation is advantageous in preventing oxidation of the expressed protein and avoiding side reactions with culture medium components, while periplasmic accumulation is advantageous in inhibiting degradation caused by intracellular proteases.

[0251] On the other hand, if the protein is secreted extracellularly by the transformant, the process of cell disruption and extraction is unnecessary, thus reducing manufacturing costs. Conversely, in the case of a host that also secretes proteases, the target protein may be degraded during culture.

[0252] As a specific method, when proteins accumulate within cultured cells (including the periplasmic region), the bacterial cells can be collected from the culture medium by methods such as centrifugation or filtration. The cells are then pulverized using methods such as ultrasonic pulverization or French press filtration, and / or dissolved by adding surfactants, thereby recovering the proteins accumulated within the cells. If proteases also dissolve at this point, the target protein may be decomposed.

[0253] When secreting recombinant proteins, after the culture is completed, the cultured cells can be separated from the supernatant containing the secreted proteins using general separation methods such as centrifugation and filtration, and the produced recombinant proteins can be recovered.

[0254] When manufacturing the proteins of the present invention using a cell-free protein synthesis system, there are no particular limitations on the cell-free protein synthesis system as long as it uses cell extracts to synthesize proteins in vitro. For example, synthesis systems derived from prokaryotic cells, plant cells, or higher animal cells can be used.

[0255] The protein of the present invention can also be manufactured by culturing the above-mentioned transformant in a culture medium to express it in the form of a protein fused with other proteins, collecting the fusion protein from the culture, cleaving the fusion protein using an appropriate protease, and collecting the desired protein.

[0256] The method for culturing the above-mentioned transformants in the culture medium is the same as the usual method used in the culture of the host. There are no particular restrictions on the culture medium used to culture the obtained transformants, as long as it can produce the protein with high efficiency and high yield.

[0257] Specifically, carbon and nitrogen sources such as glucose, sucrose, glycerol, peptone, meat extract, yeast extract, and casein amino acids can be used. In addition, inorganic salts such as potassium, sodium, phosphate, magnesium, manganese, zinc, and iron salts can be added as needed. When using auxotrophic host cells, nutrients required for growth and reproduction can be added. Furthermore, antibiotics such as penicillin, erythromycin, chloramphenicol, neomycin, and kanamycin can be added as needed.

[0258] There are no particular limitations on the culture medium used to culture transformants obtained from Escherichia coli. Examples include LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl) or 2xYT medium (1.6% tryptone, 1.0% yeast extract, 0.5% NaCl).

[0259] There are no particular limitations on the culture medium used to culture transformants obtained from Bacillus brevis bacteria as hosts. Examples include TM medium (1% peptone, 0.5% meat extract, 0.2% yeast extract, 1% glucose; pH 7.0) or 2SL medium (4% peptone, 0.5% yeast extract, 2% glucose; pH 7.2).

[0260] In addition, to inhibit the degradation and reduction of the target protein by host-derived proteases present in and outside the bacteria, various known protease inhibitors can be added at appropriate concentrations, namely, benzyl sulfonyl fluoride (PMSF), Benzamidine, 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), antiprotease, chymostatin, leupeptin, pepstatin A, phosphoramidon, aprotinin, EDTA, and / or other commercially available protease inhibitors.

[0261] Furthermore, to ensure the correct folding of the proteins of the present invention, molecular chaperones such as GroEL / ES, Hsp70 / DnaK, Hsp90, and Hsp104 / ClpB can be utilized. Molecular chaperones can coexist with the proteins of the present invention through methods such as co-expression or fusion proteinization. When the goal is correct protein folding, methods such as adding additives to the culture medium to promote correct folding and culturing at low temperatures can also be used, but are not limited to these methods.

[0262] Recombinant proteins can be produced by aerobic culture at a temperature of 15–42°C, preferably 20–37°C, under aerated and stirred conditions for several hours to several days. Depending on the specific circumstances, aerobic culture can also be performed by blocking aeration.

[0263] Purification of recombinant proteins can be performed alone or in appropriate combinations using affinity chromatography, cation or anion exchange chromatography, gel filtration chromatography, etc.

[0264] Confirmation of whether the purified substance is the target protein can be made using common methods, such as SDS-polyacrylamide gel electrophoresis, N-terminal amino acid sequence analysis, immunoblotting, enzyme-linked immunosorbent assay (ELISA), etc.

[0265] <Affinity Separator>

[0266] By immobilizing the protein of the present invention as an affinity ligand onto a support made of a water-insoluble substrate, the affinity separating agent of the present invention can be obtained. Here, "affinity ligand" refers to a substance (functional group) that selectively captures (binds) a target molecule from a set of molecules based on the specific affinity between molecules, exemplified by the binding of antigens to immunoglobulins. In this specification, it refers to a protein that specifically binds to immunoglobulins. In this specification, when abbreviated as "ligand," it also has the same meaning as "affinity ligand."

[0267] Examples of water-insoluble substrates used in this invention include inorganic supports such as glass beads and silica gel; synthetic polymers such as cross-linked polyvinyl alcohol, cross-linked polyacrylate, cross-linked polyacrylamide, and cross-linked polystyrene; organic supports composed of polysaccharides such as crystalline cellulose, cross-linked cellulose, cross-linked agarose, and cross-linked dextran; and composite supports such as organic-organic and organic-inorganic combinations thereof.

[0268] Examples of commercially available products include GCL2000 (manufactured by Biochemical Industrial Co., Ltd.) as a porous cellulose gel, Sephacryl (registered trademark) S-1000 (manufactured by GE HealthCare Japan Co., Ltd.) covalently crosslinked with allyl dextran and methylenebisacrylamide, Toyopearl (registered trademark) (manufactured by TOSOH Co., Ltd.) as an acrylate-based support, Sepharose (registered trademark) CL4B (manufactured by GE HealthCare Japan Co., Ltd.) as an agarose-based crosslinking support, and Cellufine (registered trademark) (manufactured by JNC Co., Ltd.) as a cellulose-based crosslinking support. However, the water-insoluble support of the present invention is not limited to these illustrated supports.

[0269] Furthermore, considering the purpose and method of using the affinity separating agent of the present invention, it is preferable that the water-insoluble support used in the present invention has a large surface area, and preferably a porous material with a large number of pores of appropriate size. As for the form of the support, beads, monoliths, fibers, membranes (including hollow fibers), etc., are all acceptable, and any form can be selected.

[0270] Ligand immobilization methods include, for example, utilizing the amino, carboxyl, hydroxyl, carbamate, polyvinyloxy, or thiol groups present in the ligand to bind to the support via conventional coupling methods.

[0271] As a coupling method, examples include activating the support by reacting it with cyanogen bromide, epichlorohydrin, diglycidyl ether, p-toluenesulfonyl chloride, trifluoroethanesulfonyl chloride, hydrazine, and sodium periodate (or introducing reactive functional groups onto the surface of the support), and then coupling it with a compound that acts as a ligand to achieve fixation. Alternatively, examples include adding a condensing agent such as carbodiimide or a reagent with multiple functional groups in the molecule, such as glutaraldehyde, to the system containing the support and the compound that acts as a ligand to achieve condensation and cross-linking to achieve fixation.

[0272] Alternatively, a spacer molecule composed of multiple atoms can be introduced between the ligand and the support, or the ligand can be directly immobilized on the support. Therefore, for immobilization, the protein of the present invention can be chemically modified, or amino acid residues useful for immobilization can be added.

[0273] When an amino acid residue useful for immobilization is added, the added amino acid residue is called a immobilization tag. For immobilization tags, any number of amino acid residues, more than one, can be added, and the addition positions can be the N-terminal side and / or C-terminal side of the ligand. Examples of amino acids useful for immobilization include those with functional groups in their side chains that are useful for the chemical reactions of immobilization, such as lysine (K) containing an amino group in its side chain and cysteine ​​(C) containing a thiol group in its side chain.

[0274] On the other hand, as a method for directly immobilizing ligands onto a support, one example is replacing or inserting amino acids that are useful for immobilization at any position in the amino acid sequence of the ligand.

[0275] The essence of this invention is to apply the same effects to proteins as those to the separation agent (matrix) used to immobilize the protein as a ligand, regardless of any modifications or alterations made for immobilization.

[0276] Examples of fixation methods for the ligand and support in the affinity separating agent of the present invention include multi-point fixation and single-point fixation. Multi-point fixation refers to a method in which the ligand body and / or fixation tag are fixed by chemical bonding with the support at two or more points, while single-point fixation refers to a method in which the ligand body or fixation tag is fixed by chemical bonding with the support at one point.

[0277] When a ligand is immobilized by binding to a support, the two or more domains contained in the ligand are distinguished as immobilized domains and non-immobilized domains. Here, immobilized domains refer to those that bind to the support at multiple points or a single point, while non-immobilized domains refer to those that do not bind to the support.

[0278] In the affinity separator of the present invention, in order to achieve the effect of making the ligand function stably without being affected by proteases when used in antibody purification processes, etc., it is preferable that the ligand contains one or more non-fixed structural domains, preferably two or more, and in the case of n-mer ligands, preferably n-1 or more are non-fixed structural domains, and most preferably all n are non-fixed structural domains.

[0279] The following four types can be cited as examples of fixed-domain versus non-fixed-domain patterns in ligands fixed in the support.

[0280] (1) The case where all domains in the ligand are fixed domains

[0281] (2) The case where the N-terminal and / or C-terminal domains of the ligand are one or more fixed domains.

[0282] (3) The case where the domains other than the two ends of the ligand are one or more fixed domains.

[0283] (4) In the case where a fixation tag is introduced at the N-terminus and / or C-terminus of the ligand, in the absence of a fixation domain.

[0284] The following is a schematic diagram illustrating the immobilization of tetrameric proteins as ligands on a support. Figures 2-5 Taking this as an example, the same understanding applies to proteins that are more than two-dimers. It should be noted that... Figures 2-5 In this diagram, the four domains contained in the tetramer are represented as a to d, and the amino acid sequences corresponding to the first and second linker elements are represented by solid lines connecting a to d. It should be noted that the amino acid sequences of a to d can be the same or different, and this can be understood in the same way regardless of whether a and d are C-terminal or N-terminal.

[0285] In the case described in (1) above, no non-fixed structural domain is formed. For example, Figure 2 In the schematic representation, since all domains a to d are bound to the support, the ligand can still function in its bound state when the linker is cleaved by the protease during the purification process using an affinity separator.

[0286] In the case of (2) above, there is more than one non-fixed structural domain. Figure 3 (A) to (C) are used to represent schematically. Figure 3In (A), the two domains (a, b) at the N-terminus or C-terminus of the ligand are fixed domains that bind to the carrier, while the two domains (c, d) at the opposite end are non-fixed domains that do not bind to the carrier. In this case, the ligand can still function even if the linking element between a and b is severed, but if the linking element between b and c or c and d is severed, c and d will detach from the carrier and cannot function as ligands, which is therefore not preferred.

[0287] exist Figure 3 In (B), the N-terminal or C-terminal domain (a) of the ligand is a fixed domain that binds to the carrier, while the three domains (b, c, d) connected to it are non-fixed domains that do not bind to the carrier. In this case, if any of the connecting elements—a and b, b and c, or c and d—is severed, b through d will detach from the carrier and cannot function as ligands, which is therefore undesirable. If the connecting element between the fixed and non-fixed domains, such as the one connecting a and b, is severed, the domain continuously bound to the non-fixed domain also loses its function, which is particularly problematic.

[0288] exist Figure 3 In (C), the N-terminal and C-terminal domains (a, d) of the ligand are fixed domains that bind to the carrier, while the two intermediate domains (b, c) are non-fixed domains that do not bind to the carrier. In this case, if any two or more of the connecting elements between a and b, b and c, or c and d are severed, b and c will detach from the carrier and cannot function as ligands, thus this is not preferred.

[0289] In the case of (3) above, there is more than one non-fixed structural domain. Figure 4 (A) Figure 4 (B) in the diagram is used to represent this schematically. Figure 4 In (A), a domain (c (and b) located between the N-terminus and C-terminus of the ligand is a fixed domain that binds to the carrier, while the two domains (a and d) at the two ends and the one domain connected to them (b (and c)) are non-fixed domains that do not bind to the carrier. In this case, if any of the connecting elements a and b, b and c, or c and d is severed, a, b, and d will detach from the carrier and fail to function as ligands, which is undesirable. Severing the connecting elements between fixed and non-fixed domains, such as the connecting elements b and c or c and d, is particularly problematic.

[0290] exist Figure 4In (B), the two domains (b, c) located between the N-terminus and C-terminus of the ligand are fixed domains that bind to the carrier, while the two domains (a, d) at the two ends are non-fixed domains that do not bind to the carrier. In this case, even if the linking element between b and c is severed, the ligand can still function. However, if the linking element between a and b or between c and d is severed, c and d will detach from the carrier and cannot function as ligands, which is therefore not preferred.

[0291] The above (4) case is the case where the fixed label is introduced to form a combination with the carrier, and all structural domains are non-fixed structural domains. Figure 5 (A) Figure 5 (B) in the diagram is used to represent this schematically. Figure 5 In (A), a fixation tag is introduced into the terminal sequence of the N-terminal or C-terminal domain (a) of the ligand to bind it to the carrier. At this time, regardless of which linking element is severed—a to b, b to c, or c to d—b to d detach from the carrier and cannot function as a ligand, which is therefore undesirable. If a with the fixation tag is considered the fixed domain, then the severing of the linking element between a and b is similar to the above... Figure 3 Similarly, (B) in this context becomes particularly problematic. Furthermore, if the terminal sequence of 'a' is cleaved by the protease, the entire domain is released and cannot function as a ligand, thus posing a significant challenge. Therefore, a mutation to confer protease resistance needs to be introduced into the terminal (C-terminus or N-terminus) sequence of the tag used to ligand 'a'.

[0292] exist Figure 5 In (B), a fixed tag is introduced into the terminal sequences of the N-terminal and C-terminal domains (a, d) of the ligand to bind it to the carrier. If a and d with the fixed tag are considered as fixed domains, then the above-mentioned... Figure 3 (C) The same problem. Furthermore, if the terminal sequences of a and d are cleaved by the protease, the entire domain is released and cannot function as a ligand, thus becoming particularly problematic. Therefore, it is necessary to introduce mutations to confer protease resistance into the terminal (C-terminal and N-terminal) sequences of the tag used to link and fix a and d.

[0293] As described above, the variant binding element sites in the proteins of the first, third, and second embodiments of the present invention are preferably located between fixed and non-fixed structural domains, and between non-fixed structural domains. More preferably, they are located at least between fixed and non-fixed structural domains, and even more preferably, they are located between non-fixed structural domains as well.

[0294] In addition, when introducing a fixation tag into a ligand, the domain having the fixation tag can be used as the fixation domain, and the preferred approach is considered within the above-mentioned range.

[0295] In the second manner of the first embodiment of the invention, the position of the domain (variant domain) in which the lysine at position 4 and the lysine at the C-terminus are missing or replaced is preferably at least a fixed domain, more preferably a non-fixed domain directly connected to the fixed domain is also a variant domain, and even more preferably a non-fixed domain closer to the fixed domain in a series of connected non-fixed domains is also a variant domain.

[0296] On the other hand, for the non-fixed domain furthest from the fixed domain, there is no particular problem with the end of the side not connected to other domains being affected by the protease, so it does not have to be a variant domain. However, if the furthest non-fixed domain is a variant domain, the connection position with other domains is not affected by the protease, which is preferred. In addition, when introducing a fixation tag into the ligand, the domain having the fixation tag can be used as the fixed domain, and the preferred approach is considered within the above-mentioned range.

[0297] The binding site of the ligand to the carrier is not particularly limited, but it is preferably at the end of the ligand sequence. Fixation at the end allows for greater ligand flexibility, ensuring a wider binding area to immunoglobulins and higher binding affinity. If the ligand is at the end, either the N-terminus or the C-terminus is acceptable.

[0298] The affinity separating agent of the present invention is preferably an affinity separating agent that binds to proteins containing the Fc region of immunoglobulins. Examples of proteins containing the Fc region of immunoglobulins that bind to the affinity separating agent include antibodies, antibody derivatives, antibody fragments, and antibody fragment derivatives. These proteins can be separated and purified using affinity column chromatography.

[0299] It should be noted that these proteins can generally be produced using CHO cells derived from Chinese hamster ovaries, mouse myeloma Sp2 / 0 cells, NSO cells, methanol-nutritive Pichia pastoris, baker's yeast, Aspergillus, etc. When proteases are present in their culture medium, the affinity separation agent of the present invention can minimize the degradation caused by the protease, and is therefore preferred.

[0300] Here, "antibody containing the Fc region of an immunoglobulin" can be exemplified by IgG. "Antibody derivative" refers to IgG derivatives, such as chimeric antibodies formed by replacing a portion of the structural domain of human IgG with the structural domain of IgG antibodies from other species, and humanized antibodies formed by replacing the CDR portion of human IgG with the CDR portion of antibodies from other species.

[0301] As an "antibody fragment," an example is a protein consisting solely of the Fab region of human IgG. As an "antibody fragment derivative," an example is an artificial antibody formed by fusing the Fv and Fc regions of human IgG. It should be noted that the term "antibody-like molecule" is used in the specification as a consistent name for these antibodies, antibody derivatives, antibody fragments, and antibody fragment derivatives.

[0302] The affinity separating agent of the present invention can be used to separate proteins containing the Fc region of immunoglobulins. Specifically, a liquid chromatography column comprising the affinity separating agent of the present invention and having at least one container filled with the affinity separating agent can be prepared.

[0303] The separation of proteins containing the Fc region (the aforementioned antibodies, antibody derivatives, antibody fragments, and antibody fragment derivatives) can be achieved by following an affinity column-based chromatography purification method using a commercially available Protein A column (Reference 1: Roque ACA et al., “J. Chromatogr. A”, 2007, Vol. 1160, pp. 44-55).

[0304] Specifically, after adjusting the buffer solution containing antibodies, antibody derivatives, antibody fragments, and antibody fragment derivatives to neutral, the solution is passed through the liquid chromatography column of this invention to adsorb the antibodies, antibody derivatives, antibody fragments, and antibody fragment derivatives. Next, an appropriate amount of pure buffer is passed through the liquid chromatography column to wash the inside of the column.

[0305] At this point, the desired antibody, antibody derivative, antibody fragment, and antibody fragment derivative are adsorbed by the affinity separating agent of the present invention within the column. Next, an acidic buffer (sometimes containing substances that promote dissociation from the matrix) adjusted to an appropriate pH is passed through the column, causing the desired antibody, antibody derivative, antibody fragment, and antibody fragment derivative to dissolve, achieving high-purity purification.

[0306] The buffer solution (sometimes a solution containing a suitable modifier or organic solvent) is rinsed by passing it through the affinity separator of the present invention. The affinity separator of the present invention can then be reused. The buffer solution is a suitable strong acid or strong base pure buffer solution that does not damage the ligand compound or the substrate of the support at all.

[0307] Generally, the binding of the various domains constituting protein A to the Fc region is stronger than that to the Fab region (Reference 1). Therefore, the "affinity for immunoglobulins" of the protein of the present invention essentially refers to the affinity for the Fc region; even if the binding force with only the Fab region changes, the strength of the affinity for immunoglobulins does not change significantly. For the protein of the present invention, the reduced secondary affinity (secondary affinity) of the immunoglobulin-binding domain of protein A to the Fab region effectively eliminates the influence of secondary binding (secondary binding) in the interaction with immunoglobulins.

[0308] On the other hand, since the affinity for the Fc region is maintained, the affinity for the immunoglobulin as a whole is also maintained. When determining the affinity for human immunoglobulin G preparations using the Biacore system described later, the affinity constant (KA) of the protein of the present invention for immunoglobulins is preferably 10. 6 (M －1 ) or more, preferably 10 7 (M －1 )above.

[0309] The affinity of the protein of the present invention for immunoglobulins can be measured using biosensors such as the Biacore (registered trademark) system (manufactured by GE HealthCare Japan Co., Ltd.) which employs the principle of surface plasmon resonance, but the measurement method is not limited thereto.

[0310] As for the assay conditions, as long as the binding signal when protein A binds to the Fc region of immunoglobulin can be detected, it can be easily evaluated by measuring at a temperature of 20–40°C (constant temperature) and a neutral pH of 6–8.

[0311] Examples of proteins exhibiting affinity for the present invention include, but are not limited to, immunoglobulin molecules and their derivatives that contain both Fab and Fc regions without deficiency. The proteins of the present invention also exhibit affinity for proteins containing a portion of the Fc region; the binding target need not be a protein containing the complete Fc region. Since the stereostructure of antibodies is already known, further modifications (fragmentation, etc.) to the Fab and Fc regions can be made to maintain the stereostructure of the region binding to the proteins of the present invention in protein engineering, and the proteins of the present invention can also bind to their derivatives.

[0312] In addition, the binding capacity of the affinity separating agent that fixes the protein of the present invention to immunoglobulins can be evaluated by comparing static adsorption capacity and dynamic adsorption capacity, but is not limited thereto. The static adsorption capacity evaluates how much immunoglobulin can be adsorbed by the separating agent immersed in an excess of immunoglobulin solution; the dynamic adsorption capacity evaluates the amount of solution that passes through the column filled with the separating agent when the immunoglobulin solution flows through it.

[0313] Example

[0314] The present invention will be described in more detail below with reference to embodiments, but the present invention is not limited to these embodiments.

[0315] <Example of Embodiment 1 of the present invention>

[0316] [Production example]

[0317] 1) Construct wild-type protein A expression plasmid

[0318] A chemically synthesized DNA sequence (Sequence No. 7) encoding a wild-type C-domain dimer (WT) was appended to the 5' end with catatg (NdeI) and the 3' end with ctcgag (XhoI). The obtained DNA fragment was digested using restriction endonucleases NdeI and XhoI (both manufactured by Thermo Scientific) and then purified and recovered.

[0319] It should be noted that sequence number 7 was determined based on a DNA sequence derived from Staphylococcus aureus, and was determined through codon optimization for expression in Escherichia coli.

[0320] As the protein expression vector, pET22b (Merck) was selected, which has a T7 promoter and a 6xHis tag and a stop codon downstream of the multiple cloning site. pET22b was digested with restriction endonucleases NdeI and XhoI (both ThermoScientific) and then purified and recovered.

[0321] The restriction endonuclease-treated DNA fragment at sequence number 7 was ligated with the pET22b vector using a DNA ligase (LigaFast DNA Rapid Ligation System, Promega) to construct the WT expression vector.

[0322] Escherichia coli JM109 (manufactured by TaKaRa) was transformed using the WT expression vector, and plasmid DNA was amplified and extracted using conventional methods.

[0323] 2) Constructing a protein A variant (Mut) expression plasmid

[0324] Expression vectors containing DNA sequences (sequence numbers 34–46) encoding proteins Mut1–Mut13 with partial deletions and / or substitutions of the wild-type C-domain dimer amino acid sequence were constructed. The Mut1–13 expression vectors were prepared using the templates shown in Table 1 and the synthetic oligonucleotide primer set (sequence numbers 8–33) via a rapid mutagenesis assay (QuikChange site-directed mutagenesis kit, Agilent Technologies). The rapid mutagenesis assay was performed according to Agilent Technologies' procedures.

[0325] Table 1

[0326]

[0327] The obtained Mut1-13 expression vectors were amplified and extracted using conventional methods after transformation of JM109 (manufactured by TaKaRa).

[0328] The DNA sequences of the Mut1–13 expression vectors were analyzed using a 3130xl DNA sequencer (Applied Biosystems). Sequencing PCR reactions for the expression vectors were performed using the Big DyeTerminator v.1.1 cycle sequencing kit (Applied Biosystems) according to the attached protocol. The obtained sequencing PCR products were purified using standard methods for DNA sequence analysis.

[0329] The DNA sequences encoding the expressed proteins in the Mut1-13 expression vectors obtained in this embodiment are shown in sequence numbers 34-46.

[0330] 3) Protein expression

[0331] The Rosetta(DE3) (Merck) vectors obtained in 1) and 2) above were used to transform the target proteins WT and Mut1-13, respectively, to obtain transformants. The transformation method was based on Merck's procedures.

[0332] Transformants expressing the target protein were inoculated into LB medium containing 50 mg / L carbenicillin and cultured overnight at 30°C to obtain a pre-culture. 10 mL of the pre-culture was then inoculated into 500 mL of LB medium (containing 50 mg / L carbenicillin) and cultured at 30°C and 130 rpm until OD600 ≈ 0.6–0.8. Isopropyl 1-thio-β-D-galactoside (IPTG) was added at a final concentration of 0.1 mM, and the culture was continued for another 4 hours. After incubation, the bacteria were collected by centrifugation.

[0333] 4) Protein recovery

[0334] The bacterial cells collected in step 3) above were suspended in 50 mL of suspension buffer (50 mM imidazole, pH 8.0, 500 mM NaCl) and ultrasonically pulverized. Centrifugation was performed to separate the supernatant and precipitate. The supernatant was fed to a HisTrap HP 5 mL column (GE HealthCare Japan) equilibrated with suspension buffer. After washing with equilibration buffer, the target protein was dissolved using dissolution buffer (175 mM imidazole, pH 8.0, 500 mM NaCl). The dissolved target protein was replaced with desalted water by dialysis. The target protein was concentrated to approximately 30–40 mg / mL using an Amicon-Ultra 10K (Merck Millipore) centrifugation. The concentrated target protein was then diluted to 2 mg / mL using PBS buffer.

[0335] The purified target protein was fed into Tricine SDS-PAGE (e-PAGEL R15S; manufactured by ATTO), and a single band was observed at a molecular weight of approximately 14,000 Da. The target protein obtained using the WT expression vector was a protein with amino acid sequence number 47, while the target proteins obtained using the Mut1–13 expression vectors were proteins with amino acid sequences numbered 48–60, respectively.

[0336] Table 2 shows the sequences near the linker position (first linker element) of WT and the sequences near the variant linker elements of Mut1–13. In the dimer of the C domain, the amino acid at position D58 is the C-terminus of the first domain, and the amino acid at position D1' is the N-terminus of the second domain. Additionally, " / " (slash) indicates the deletion of an amino acid.

[0337] Table 2

[0338]

[0339] [Example 1-1]

[0340] <Evaluation of Trypsin Tolerance>

[0341] 1) Evaluate tolerance to trypsin, a protease, using the following methods.

[0342] Mix the Mut1 protein obtained in the preparation example with trypsin and heat at 37°C for 15 minutes. The dosage is as follows.

[0343] • Prepare 10 μL of the protein from Example 1 (2 mg / mL, phosphate buffer).

[0344] • Dilution buffer (500mM, Tris-HCl, pH 8.0) 2μL

[0345] 6μL of pure water

[0346] · Trypsin solution (1 mg / mL) 2 μL

[0347] Next, 20 μL of electrophoresis diluent (200 mM Tris-HCl, pH 6.8, 200 mM DTT, 20% glycerol, 4% SDS, 0.012% bromophenol blue; 2x sample buffer) was added to the resulting mixture. 10 μL of this mixture was taken as a sample and electrophoresed using an SDS-polyacrylamide gel. For comparison, the same volume of the mixture without the added thermophilic protease was electrophoresed on the same gel.

[0348] After electrophoresis, the gel is stained, and the staining intensity is compared to evaluate whether protein degradation occurred before and after trypsin treatment. Staining intensity refers to the position and intensity of the staining bands obtained by electrophoresis.

[0349] Compared to before trypsin treatment, proteins that were almost completely decomposed (staining a staining degree of 90% or higher) were marked with ◎, proteins that were poorly decomposed (staining a staining degree of 70% or higher but lower than 90%) were marked with ○, proteins that were extensively decomposed (staining a staining degree of 30% or higher but lower than 70%) were marked with △, and proteins that were almost completely decomposed (staining a staining degree of less than 30%) were marked with ×.

[0350] The results are shown in Table 3.

[0351] 2) The following methods were used to evaluate tolerance to plasmin, which is a protease.

[0352] The trypsin solution (1 mg / mL) was replaced with the plasmin solution (10 mg / mL), and the evaluation was performed in the same manner as for the evaluation of trypsin tolerance. The results are shown in Table 3.

[0353] <Evaluation of Immunoglobulin Adsorption Capacity (SBC)>

[0354] The protein of Mut1 obtained in the 3 mg preparation example was immobilized on 1 mL of epoxy-activated porous acrylic beads to prepare a separating agent, and its binding ability with immunoglobulins was evaluated.

[0355] The static adsorption capacity of the separating agent was evaluated using standard methods. The results are shown in Table 3.

[0356] [Examples 1-2 to 1-10, Comparative Examples 1-1 to 1-4]

[0357] Instead of Mut1, Mut2 to 10 obtained in the preparation examples were used in Examples 1-2 to 1-10, WT was used in Comparative Example 1-1, and Mut11 to 13 were used in Comparative Examples 1-2 to 1-4. Otherwise, the protease resistance evaluation and immunoglobulin adsorption capacity evaluation were performed in the same manner as in Example 1-1.

[0358] The results are shown in Table 3.

[0359] Table 3

[0360]

[0361] Comparative Examples 1-1 to 1-4, in which the C-terminal lysine residues were neither deleted nor replaced, showed low trypsin resistance and plasmin resistance. In contrast, Examples 1-1 to 1-10, in which the C-terminal lysine residues were deleted or replaced, showed at least higher trypsin resistance and plasmin resistance. The linker sequence (first linker element) in Examples 1-2 and 1-3 was shorter, consisting of two amino acid residues, and further improved trypsin resistance.

[0362] <Example of Embodiment 2 of the Invention>

[0363] [Production example]

[0364] 1) Construct wild-type protein A expression plasmid

[0365] A chemically synthesized DNA sequence (Sequence No. 61) encoding a wild-type C-domain dimer (WT) was appended to the 5' end with catatg (NdeI) and the 3' end with ctcgag (XhoI). The obtained DNA fragment was digested using restriction endonucleases NdeI and XhoI (both manufactured by Thermo Scientific) and then purified and recovered.

[0366] It should be noted that sequence number 61 was determined based on a DNA sequence derived from Staphylococcus aureus, and was determined through codon optimization for expression in Escherichia coli.

[0367] As the protein expression vector, pET22b (Merck) was selected, which has a T7 promoter and a 6xHis tag and a stop codon downstream of the multiple cloning site. pET22b was digested with restriction endonucleases NdeI and XhoI (both ThermoScientific) and then purified and recovered.

[0368] The DNA fragment with restriction endonuclease-treated sequence number 61 was ligated with the pET22b vector using DNA ligase (LigaFast DNA Rapid Ligation System, Promega) to construct the WT expression vector.

[0369] Escherichia coli JM109 (manufactured by TaKaRa) was transformed using the WT expression vector, and plasmid DNA was amplified and extracted using conventional methods.

[0370] 2) Constructing a protein A variant (Mut) expression plasmid

[0371] Expression vectors containing DNA sequences (accessions 90-103) encoding proteins Mut1-Mut14 with partial deletions and / or substitutions of the wild-type C-domain dimer amino acid sequence were constructed. The Mut1-14 expression vectors were prepared using the templates shown in Table 4 and the synthetic oligonucleotide primer set (accessions 62-89) via a rapid mutagenesis assay (QuikChange site-directed mutagenesis kit, Agilent Technologies). The rapid mutagenesis assay was performed according to Agilent Technologies' protocol.

[0372] Table 4

[0373]

[0374] The obtained Mut1-14 expression vectors were obtained by transforming JM109 (manufactured by TaKaRa) and then amplifying and extracting them using conventional methods.

[0375] The DNA sequences of the obtained Mut1–14 expression vectors were analyzed using a 3130xl DNA sequencer (Applied Biosystems). Sequencing PCR reactions for the expression vectors were performed using the Big Dye Terminator v.1.1 Cyclic Sequencing Kit (Applied Biosystems) according to the accompanying protocol. The obtained sequencing PCR products were purified using standard methods for DNA sequence analysis.

[0376] The DNA sequences encoding the expressed proteins in the Mut1-14 expression vectors obtained in this embodiment are shown in sequence numbers 90-103.

[0377] 3) Protein expression

[0378] Rosetta(DE3) (Merck) was transformed using the WT and Mut1-14 expression vectors obtained in 1) and 2) above to obtain transformants expressing the target proteins WT and Mut1-14, respectively. The transformation method followed Merck's procedures.

[0379] Transformants expressing the target protein were inoculated into LB medium containing 50 mg / L carbenicillin and cultured overnight at 30°C to obtain a pre-culture. 10 mL of the pre-culture was then inoculated into 500 mL of LB medium (containing 50 mg / L carbenicillin) and cultured at 30°C and 130 rpm until OD600 ≈ 0.6–0.8. Isopropyl 1-thio-β-D-galactoside (IPTG) was added at a final concentration of 0.1 mM, and the culture was continued for another 4 hours. After incubation, the bacteria were collected by centrifugation.

[0380] 4) Protein recovery

[0381] The bacterial cells collected in step 3) above were suspended in 50 mL of suspension buffer (50 mM imidazole, pH 8.0, 500 mM NaCl) and ultrasonically pulverized. Centrifugation was performed to separate the supernatant and precipitate. The supernatant was fed to a HisTrap HP 5 mL column (GE HealthCare Japan) equilibrated with suspension buffer. After washing with equilibration buffer, the target protein was dissolved using dissolution buffer (175 mM imidazole, pH 8.0, 500 mM NaCl). The dissolved target protein was replaced with desalted water by dialysis. The target protein was concentrated to approximately 30–40 mg / mL using an Amicon-Ultra 10K (Merck Millipore) centrifugation. The concentrated target protein was then diluted to 2 mg / mL using PBS buffer.

[0382] The purified target protein was fed into Tricine SDS-PAGE (e-PAGEL R15S; manufactured by ATTO), and a single band was observed at a molecular weight of approximately 14,000 Da. The target protein obtained using the WT expression vector was a protein with amino acid sequence number 104, while the target proteins obtained using the Mut1–14 expression vectors were proteins with amino acid sequences numbered 105–118, respectively.

[0383] The sequences near the linker element (second linker element) of WT and the sequences near the variant linker elements (second linker elements) of Mut1–14 are shown in Table 5. In the dimer of the C domain, the amino acid at position D58 is the C-terminus of the first domain, and the amino acid at position D1' is the N-terminus of the second domain. Additionally, " / " (slash) indicates the deletion of an amino acid.

[0384] Table 5

[0385]

[0386] [Example 2-1]

[0387] <Protein Tolerance Evaluation>

[0388] 1) The following methods were used to evaluate tolerance to thermophilic bacterial proteases.

[0389] Mix the Mut11 protein obtained in the preparation example with the thermophilic protease and heat at 37°C for 15 minutes. The proportions are as follows.

[0390] • Prepare the protein for Example 1 (2 mg / mL, phosphate buffer) 10 μL

[0391] • Dilution buffer (500mM Tris-HCl, pH 8.0, 5mM CaCl2) 2μL

[0392] 6μL of pure water

[0393] Thermophilic bacteria protease solution (1 mg / mL) 2 μL

[0394] Next, 20 μL of electrophoresis diluent (200 mM Tris-HCl, pH 6.8, 200 mM DTT, 20% glycerol, 4% SDS, 0.012% bromophenol blue; 2x sample buffer) was added to the resulting mixture. 10 μL of this mixture was taken as a sample and electrophoresed using an SDS-polyacrylamide gel. For comparison, the same volume of the mixture without the added thermophilic protease was electrophoresed on the same gel.

[0395] After electrophoresis, the gel is stained, and the staining intensity is compared to evaluate whether protein degradation occurred before and after treatment with thermophilic proteases. Staining intensity refers to the position and intensity of the staining bands obtained by electrophoresis.

[0396] Compared to before treatment with thermophilic protease, proteins that were almost completely degraded (staining a staining degree of 90% or higher) were marked with ◎, proteins that were poorly degraded (staining a staining degree of 70% or higher but lower than 90%) were marked with ○, proteins that were heavily degraded (staining a staining degree of 30% or higher but lower than 70%) were marked with △, and proteins that were almost completely degraded (staining a staining degree of less than 30%) were marked with ×.

[0397] The results are shown in Table 6.

[0398] 2) Evaluate tolerance to trypsin, a protease, using the following methods.

[0399] The thermophilic protease solution (1 mg / mL) was changed to trypsin solution (1 mg / mL), and otherwise evaluated in the same manner as for the thermophilic protease tolerance assessment. The results are shown in Table 6.

[0400] 3) The following methods were used to evaluate tolerance to plasmin, which is a protease.

[0401] The thermophilic protease solution (1 mg / mL) was changed to a plasmin solution (10 mg / mL), and otherwise the evaluation was performed in the same manner as for the evaluation of thermophilic protease tolerance. The results are shown in Table 6.

[0402] <Evaluation of Immunoglobulin Adsorption Capacity (SBC)>

[0403] An affinity separating agent was prepared by immobilizing 3 mg of Mut11 protein obtained in the preparation example onto 1 mL of epoxy-activated porous acrylic beads. The binding affinity to immunoglobulins was evaluated. The static adsorption capacity of the separating agent was evaluated using standard methods.

[0404] The results are shown in Table 6.

[0405] [Examples 2-2 to 2-11, Comparative Examples 2-1 to 2-4]

[0406] Instead of Mut11, Mut2-10 and 13 obtained in the preparation examples were used in Examples 2-2 to 2-11, WT was used in Comparative Example 2-1, and Mut12, 14 and 15 were used in Comparative Examples 2-2 to 2-4. Otherwise, the protease resistance evaluation and immunoglobulin adsorption capacity evaluation were performed in the same manner as in Example 2-1.

[0407] The results are shown in Table 6.

[0408] Table 6

[0409]

[0410] Comparative Example 2-1, where the linker element (second linker element) was unchanged, exhibited low resistance to thermophilic proteases. In contrast, Examples 2-1 to 2-11, which had a modified linker element (modified second linker element), showed high resistance to thermophilic proteases and maintained high binding capacity. In Comparative Examples 2-2 and 2-3, although the hydrophobic amino acid at position 1 of the linker element (second linker element) was missing, the presence of a hydrophobic amino acid at position 5 resulted in low resistance to thermophilic proteases. In Comparative Example 2-4, the absence of hydrophobic amino acids at positions 1 and 5 of the linker element (second linker element) resulted in high resistance to thermophilic proteases, but the binding capacity was reduced because the number of amino acids in the linker element (second linker element) was less than one.

[0411] The present invention has been described in detail using specific methods, but those skilled in the art will understand that various changes and modifications can be made without departing from the intent and scope of the invention. Furthermore, this application incorporates the entire contents of Japanese Patent Application No. 2015-021577 and Japanese Patent Application No. 2015-021578, both filed on February 5, 2015.

[0412] Industrial availability

[0413] According to the present invention, proteins that are resistant to proteases, particularly serine proteases and thermophilic proteases, and have the ability to bind to immunoglobulins can be provided. Therefore, the proteins of the present invention can be used in the manufacture of affinity ligands with excellent binding ability to immunoglobulins or affinity separators with excellent durability.

[0414] Symbol Explanation

[0415] 1. Structural Domain

[0416] 2. The first or second connecting element

[0417] 3. Carrier surface

[0418] 4. Fixing labels

Claims

1. A protein with affinity for immunoglobulins, characterized in that, It consists of two domains derived from the C domain of protein A as described in sequence number 5, and the amino acid sequence is shown in sequence numbers 48-57.

2. A protein with affinity for immunoglobulins, characterized in that, It consists of two domains derived from the C domain of protein A as described in sequence number 5, and the amino acid sequence is shown in sequence numbers 105-115.

3. An affinity separating agent, characterized in that, The protein described in claim 1 or 2 is used as an affinity ligand and immobilized on a support made of a water-insoluble substrate.

4. A liquid chromatography column, characterized in that, The device includes the affinity separating agent as described in claim 3, and comprises at least one container filled with the affinity separating agent.

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