JP4179517B2 - Immunoglobulin affinity ligand - Google Patents

Description

  The present invention relates to a protein having affinity for immunoglobulin, and more particularly to a modified protein of an immunoglobulin binding protein having improved properties as an affinity ligand for affinity chromatography. The present invention also relates to the use of the modified protein as an immunoglobulin affinity ligand, as well as to an affinity separation matrix.

[Definition]
In the present invention, the “protein” includes any molecule having a peptide structure, and includes a partial fragment of a natural protein or a variant obtained by artificially modifying a natural sequence. An “immunoglobulin binding domain” refers to a functional unit of a polypeptide having immunoglobulin binding activity alone, and an “immunoglobulin binding protein” is a protein having a specific affinity for immunoglobulin, A protein comprising an “immunoglobulin binding domain” that non-covalently binds to globulin.

  “Functional variants” of immunoglobulin binding proteins are immunoglobulin binding proteins that have been altered by partial amino acid addition, deletion, substitution, chemical modification of amino acid residues, etc. It means that it has 80% or more homology with the previous amino acid sequence, preferably 90% or more, and can be treated as equivalent to the protein before modification in functional properties including immunoglobulin binding activity. .

“Affinity ligand” refers to a molecule that has the property of binding to a specific molecule with specific affinity. In the present invention, it refers to an immunoglobulin-binding protein that can selectively bind an immunoglobulin. In addition, it may only be called "ligand". “Affinity chromatography support” refers to a support in which an affinity ligand is immobilized on an insoluble support. Immobilization refers to covalent binding of an affinity ligand to an insoluble carrier, whereby the insoluble carrier becomes an affinity adsorbent for a target molecule (immunoglobulin).
In addition, when the ligand is immobilized on an insoluble carrier, the “orientation for maintaining affinity for immunoglobulin” is improved in the direction in which the ligand molecule can bind to the immunoglobulin in the immobilization reaction to the insoluble carrier. This means that the rate of immobilization is increased, and as a result, the amount of immunoglobulin that can be bound per unit molecule of the immobilized ligand is increased.

  In recent years, there is a high demand for highly pure protein reagents and protein pharmaceuticals in research related to biotechnology and the pharmaceutical industry. As a method for producing high-purity protein, purification processes based on various principles such as hydrophobic chromatography, gel filtration chromatography, and ion exchange chromatography are adopted. Among them, affinity chromatography separates the target protein and impurities, It is an excellent method with extremely high efficiency for concentrating the target protein. In affinity chromatography, a target protein is adsorbed on an insoluble carrier in which a molecule having specific affinity for the target protein is bound as a ligand to be separated from contaminants. Proteins are often used as the ligand molecule.

  Protein A is a protein derived from Staphylococcus aureus and has a repeating structure of five immunoglobulin binding domains having homology called E domain, D domain, A domain, B domain and C domain. It is known that only one immunoglobulin-binding domain binds to immunoglobulin (Non-patent Document 1), and the immunoglobulin-binding domain in which the amino acid sequence is partially modified along with natural protein A Recombinant proteins consisting only of these are also widely used as affinity ligands for affinity chromatography. Staphylococcus is known to express a protein having a property of binding to immunoglobulin in addition to protein A (Patent Document 1), and protein A immunoglobulin binding is included in the partial structure of this protein. A domain and a region having about 45% homology in the amino acid sequence are included. However, the usefulness of this immunoglobulin binding domain as an affinity ligand, such as chemical stability under acidic and alkaline pH conditions, is unknown.

With respect to protein A, it is an object to provide an adsorption medium having specificity and binding capacity for binding to IgG, and chemical stability at least as good as or better than other adsorbents based on natural protein A. In addition, a technique has been proposed in which protein A (rProtein A cys) into which one cysteine residue is introduced is used as a ligand, and this is immobilized at one point on an affinity carrier via a thioester bond (Patent Document 2). . In addition, while solving the problems of the chemical stability of the immobilization site and the sterilization / washing process derived therefrom, which are the problems of affinity carriers for antibody purification typified by affinity chromatography carriers with immobilized Protein A cys, etc. In order to achieve a higher amount of adsorption of antibody molecules, a technique of selectively immobilizing a C-terminal carboxyl group via an amide bond on a carrier into which a polymer compound having a primary amino group has been introduced (Patent Document 3) Has been proposed.
Furthermore, in Patent Document 4, for example, an immunoglobulin-binding protein that can be derived from a protein that can bind to a region other than the complementarity determining region (CDR) of an immunoglobulin molecule, such as the B domain of protein A of Staphylococcus. An immunoglobulin-binding protein in which one or more asparagine residues are mutated to amino acids other than glutamine or aspartic acid, and the chemical stability in the pH range of about 13 to 14 or less is improved by the mutation compared to the parent molecule A matrix for affinity separation in which the protein is bound to a solid support using the protein as a ligand has been proposed, and the best embodiment that contributes to imparting alkali resistance shown therein is the substitution of asparagine at position 23 with threonine and the substitution. Combination of asparagine at position 43 with glutamic acid Is Align.

US Pat. No. 6,654,639B1 US Patent No. US6399750B1 (see Special Table 2000-500649) JP-A-2005-112827 International Publication No. WO03 / 080655 (see Special Table 2005-538693) Nilsson B. et al., Protein engineering, 1987, Vol. 1, No. 2, pp. 107-113

  In addition to the properties required for general chromatography carriers such as high flow rate and hardness that can withstand the weight of the gel, desirable properties required for affinity chromatography carriers used in the production of immunoglobulins as antibody drug raw materials include: a) Large amount of immunoglobulin binding per unit weight or volume of carrier, (b) Little leakage of ligand in the elution process of immunoglobulin under acidic pH conditions, (c) Line washing under alkaline pH conditions The decrease in immunoglobulin binding activity in the process is small, and (d) it can be produced at low cost. In particular, the property (a) is important for reducing the purification cost of immunoglobulin. In order to solve these problems, it is of course important to select an appropriate insoluble carrier material and a method for producing a ligand protein. In addition, it is extremely important to improve the immunoglobulin-binding protein used as a ligand. It is.

  Various studies have been made to improve the properties (a) to (d) above. Examples of modification by modification of the ligand protein itself include improvements in (a) and (c). The common point of the technologies (Patent Documents 2 and 3) developed for the purpose of producing a carrier excellent in the properties of (a) is that the ligand protein is immobilized on the carrier at one point in the vicinity of the C-terminus, In both cases, there remains a problem that the ligand protein or fragment thereof that has been cleaved during the elution of immunoglobulin under acidic pH conditions tends to leak. In addition, in order to prepare a carrier having excellent properties of (b), it is known that a method of immobilizing a ligand protein on a carrier at multiple points is effective contrary to (a), and a protein A molecule It is generally performed to fix to a carrier via a plurality of primary amino groups present therein. However, when immobilized by this method, the orientation of the ligand protein cannot be controlled, and the immobilized ligand protein includes molecules that cannot bind to immunoglobulin, so the amount of immunoglobulin bound per unit weight or volume of the carrier is descend. That is, in the existing technology, (a) and (b) are mutually contradictory factors, and at the same time, new technology development for solving both of them is desired. Furthermore, what is shown in the disclosed technique (Patent Document 4) for producing a carrier excellent in the properties of (c) is still insufficient in chemical stability. Further, protein A protein produced mainly by gene recombination techniques is used for immunoglobulin affinity chromatography carriers that are currently available on the market, but improvements in the production method are still desired.

  Therefore, the present invention has improved properties as an affinity ligand for affinity chromatography in immunoglobulin binding proteins, that is, in addition to the properties of (a) and (b) which are mutually contradictory, An object is to provide an excellent modified protein. The present invention also relates to the use of the modified protein as an immunoglobulin affinity ligand and an affinity separation matrix, that is, an immunoglobulin that combines high immunoglobulin binding ability and chemical stability and can be produced at low cost. It is an object of the present invention to provide a carrier for affinity chromatography.

  When a ligand protein is immobilized on an affinity chromatography support and a single-point cross-linking method that allows easy control of orientation is employed, the ligand protein or peptide fragment is likely to leak due to cleavage of the cross-linked moiety or peptide chain. On the other hand, when a protein A immunoglobulin binding domain protein, which is normally used, is used as a ligand as it is and fixed to a carrier at multiple points, leakage of the ligand protein is less likely to occur, but the orientation of the ligand cannot be controlled, so the unit carrier The amount of immunoglobulin binding per unit is low. The inventors of the present invention attempted to make the above conflicting factors compatible by modifying the ligand protein so that the orientation of cross-linking can be controlled even if it is fixed to the carrier at multiple points.

  In one protein A protein derived from Staphylococcus, there are five immunoglobulin-binding domains as described above, and each of these immunoglobulin-binding domains has three α-helix structures and each other. It is composed of two connected loop structures. All 10 amino acids thought to be directly involved in binding to the Fc region of immunoglobulin are present on the first helix (helix 1) and the second helix (helix 2) from the N-terminal side. (Tashiro M. et al. 1997 High-resolution solution NMR structure of the Z domain of staphyrococcal protein A, Journal of Molecular Biology Vol.272, No.272, pp.573-590). Furthermore, the immunoglobulin binding domain can bind to immunoglobulin even in the absence of helix 3 (Melissa A. et al. 1997, Structural mimicry of a native protein by a minimized binding domain. Proceeding Natural Academy of Sciences USA, 94, pp. 10080-0085).

  In reactions widely used for immobilizing proteins on affinity chromatography carriers, the main active groups on the protein side that can be used for binding reactions with carriers are cysteine thiol groups, N-terminal amino acids and lysine amino groups, and C-terminals. Amino acid, glutamic acid, carboxyl group of aspartic acid and the like. The present inventors paid attention to lysine residues as amino acid residues advantageous for binding to a carrier at multiple points while controlling the orientation, and based on the three-dimensional structure data of the immunoglobulin A binding domain of protein A The arrangement of lysine within was designed. Specifically, the number of lysine residues present on the protein surface of helix 1 and helix 2, which are immunoglobulin binding regions, is reduced as much as possible, and lysine residues as much as possible on helix 3 and the surrounding protein surface The amino acid substitution which increases the number was examined. In addition, the present inventors have made a substitution to eliminate the aspartate-proline sequence, which is considered to be susceptible to cleavage under acidic pH conditions, in order to prevent leakage of ligand proteins during the elution of immunoglobulins under acidic pH conditions. Was examined.

  The present inventors evaluated the behavior of the prepared modified protein in cation exchange chromatography, cleavage characteristics under acidic pH conditions, immunoglobulin binding activity when immobilized on a chromatographic support, and stability under alkaline pH conditions. As a result of repeated search for ligand proteins that are superior in many aspects, the ligand protein itself can be easily purified, and it exhibits high immunoglobulin binding activity even when immobilized on a carrier at multiple points, under acidic and alkaline pH conditions. The present inventors have succeeded in producing a very stable immunoglobulin-binding protein variant and completed the present invention.

That is, the gist of the present invention is a modified protein in the following isofunctional variant of the immunoglobulin binding protein of (1) to (9).
(1) An immunoglobulin-binding protein capable of binding to a region other than the complementarity determining region (CDR) of an immunoglobulin molecule, which is a C domain of Staphylococcus protein A defined by SEQ ID NO: 1. The ratio of the number of lysine after position 39 to the number of lysine at position (A) 1-38 of the variant or the Z domain defined by SEQ ID NO: 2 is higher than that of the molecule before modification. When immobilized on an insoluble carrier via its own amino group, the orientation for maintaining affinity for immunoglobulin is improved compared to the molecule before modification and / or (B) aspartic acid -By eliminating the proline sequence, the chemical stability under acidic pH conditions is improved compared to the molecule before modification. Protein or its equivalent functional variant.
(1) is the immunoglobulin binding protein represented by SEQ ID NO: 1 or SEQ ID NO: 2 or an equivalent functional variant thereof, wherein (A) the ratio of the number of lysine after position 39 to the number of lysine at position 1-38 is not changed When the protein is immobilized on an insoluble carrier via its own amino group, the orientation for maintaining affinity for immunoglobulin is higher than that of the molecule before modification. A modified protein embodiment characterized in that it is improved, (B) by eliminating the aspartic acid-proline sequence, chemical stability under acidic pH conditions is improved compared to the molecule before modification. And (A) the ratio of the number of lysine after position 39 to the number of lysine at positions 1-38 is higher than that of the molecule before modification. Thus, when the protein is immobilized on an insoluble carrier via its own amino group, the orientation for maintaining affinity for immunoglobulin is improved compared to the molecule before modification, and ( B) By including the sequence of aspartic acid-proline, the chemical stability under acidic pH conditions is improved as compared with the molecule before modification, and the modified protein embodiment is included.

(2) In the above (A), substitution with lysine after position 39 and / or modification to add lysine, and / or substitution of lysine originally in positions 4, 7, and 35 with other amino acids The modified protein according to (1) or an equivalent functional variant thereof by modification.
In the embodiment including (A) in (1) above, the above (2) is substitution to lysine after position 39, addition of lysine, lysine originally present at positions 4, 7, and 35 to other amino acids. Substitution, substitution to lysine after position 39 and addition of lysine, substitution to lysine after position 39 and substitution of other lysine from the original lysine at positions 4, 7, and 35, addition to lysine after position 39 Including embodiments of substitution, addition of lysine, substitution of lysine originally in positions 4, 7, 35 to other amino acids, addition of lysine and substitution of lysine originally in positions 4, 7, 35 to other amino acids .

(3) The substitution to lysine after the 39th position is that 1 to 6 of the amino acids at the 40th, 43th, 46th, 53th, 54th and 56th positions are substituted with lysine. The modified protein according to (2) or an equivalent functional variant thereof.
(4) Modification of substitution of lysine originally in positions 4, 7, and 35 with other amino acids is such that amino acids at positions 4, 7 and 35 are alanine, glutamine, asparagine, valine, serine, threonine, histidine and tyrosine. Or the altered protein according to (2) or an equivalent functional variant thereof, which is substituted with arginine.
(5) The modified protein according to (2) or an equivalent functional variant thereof, wherein the modification to add lysine is modification to add 1 to 5 lysines to the C-terminus.

(6) The modified protein according to (1), wherein (B) is a modification in which aspartic acid at position 37 is substituted with an amino acid other than aspartic acid, or a modification in which proline at position 38 is substituted with an amino acid other than proline; Its isofunctional variant.
(6) is an embodiment including (B) of (1) above, a modification of substituting aspartic acid at position 37 with an amino acid other than aspartic acid, a modification of substituting proline at position 38 with an amino acid other than proline, It includes a modification in which aspartic acid at position 37 is substituted with an amino acid other than aspartic acid and a modification in which the proline at position 38 is substituted with an amino acid other than proline.

(7) The modification in which aspartic acid at position 37 is substituted with an amino acid other than aspartic acid is that the amino acid at position 37 is substituted with alanine, glutamic acid, serine, threonine, leucine, or isoleucine. The described modified protein or an equivalent functional variant thereof.
(8) The modified protein according to (6) or an equivalent function thereof, wherein the modification of substituting proline at position 38 with an amino acid other than proline is that the amino acid at position 38 is substituted with alanine, serine, or threonine. Mutant.

(9) The modified protein or the equivalent functional variant thereof according to (1), comprising the amino acid sequence defined by SEQ ID NO: 3.

Moreover, this invention makes the summary the following multimer of (10).
(10) Two to five immunoglobulin-binding protein units are linked, and the modified protein according to any one of (1) to (9) or an equivalent functional variant thereof is included therein Multimer to do.

The gist of the present invention is the following nucleic acid (11).
(11) A nucleic acid encoding the modified protein according to any one of (1) to (10) or an equivalent functional variant thereof, or a multimer containing them.
The “multimer containing them” means “multimers containing a modified protein or an equivalent functional variant thereof”.

The gist of the present invention is the following gene expression system (12).
(12) A gene expression system comprising the nucleic acid according to (11).

The gist of the present invention is the following carrier for affinity chromatography (13).
(13) A carrier for affinity chromatography comprising the modified protein according to any one of (1) to (10) or an equivalent functional variant thereof, or a multimer containing them as an affinity ligand.

The gist of the present invention is the following affinity column (14).
(14) An affinity column comprising the affinity chromatography carrier according to (13).

The gist of the present invention is the following affinity separation method (15).
(15) An affinity separation method for IgG, IgA and / or IgM, wherein the affinity column according to (14) is used.

Furthermore, the gist of the present invention is the following protein chip (16).
(16) A protein chip comprising the modified protein according to any one of (1) to (10) or an equivalent functional variant thereof, or a multimer containing them.

According to the present invention, a carrier for affinity chromatography having high immunoglobulin binding ability, chemically stable and less leakage of ligand protein can be provided at low cost. More specifically, in the modified domain of protein A C domain or the Z domain defined by SEQ ID NO: 2, the ratio of the number of lysine after position 39 to the number of lysine at position 1-38 is increased compared to the molecule before modification. By modifying the protein, the protein can be immobilized on an insoluble carrier selectively through the amino group at the 39th and subsequent positions, so the orientation as an affinity ligand for immunoglobulin is higher than that of the molecule before modification. By improving, (a) an affinity carrier having a large amount of immunoglobulin binding per unit weight or volume of the carrier can be provided (described in Example 7). In addition, by such modification, the protein can be immobilized on an insoluble carrier via a plurality of amino groups, and in addition, by removing the aspartic acid-proline sequence, (b) chemical stability under acidic pH conditions (implementation) An affinity carrier that combines chemical stability (described in Example 8) under alkaline pH conditions (described in Example 3) and (c) can be obtained. Further, as the positive charge on the protein surface increases with the modification of the present invention, the protein can be easily purified by cation exchange chromatography, and (d) a ligand protein that can be produced at low cost can be provided (Example 2). Described in).
Furthermore, by using the modified protein as an immunoglobulin affinity ligand, it is possible to perform affinity purification of an immunoglobulin having high immunoglobulin binding ability and chemical stability, and the present invention includes an immunoglobulin. Products can be provided at low cost.

As a standard technique for producing the immunoglobulin binding protein of the present invention, a known gene recombination technique described in, for example, Current Protocols In Molecular Biology by Frederick M. Ausbel et al. Can be used. That is, by transforming an expression vector containing a nucleic acid sequence encoding a target modified protein into a host such as Escherichia coli and culturing the cells in an appropriate liquid medium, the cells can be produced in a larger amount and more economically than the cultured cells. Can be acquired. Specifically, since one immunoglobulin binding domain of protein A is a small protein consisting of about 60 amino acids, for example, DNA encoding a desired amino acid sequence is divided into synthetic oligonucleotides consisting of several tens of bases. The target expression vector can be obtained by synthesizing and ligating them by a ligation reaction with DNA ligase and inserting them into a plasmid vector. At that time, in order to efficiently express the protein in Escherichia coli, a nucleic acid sequence using an optimal codon of Escherichia coli is generally used by those skilled in the art. As the amino acid sequence of the immunoglobulin-binding domain before modification, any of the domains of protein A may be adopted, but among the five domains, the C domain having many lysine residues after the 39th position is used. Preferably, a Z domain sequence that has been used as an affinity ligand for immunoglobulin may be used, but substitution of glycine at position 29, which is already known to increase chemical stability, with alanine (non-patented) Most preferably, the C domain sequence (shown in SEQ ID NO: 1 in the sequence listing) subjected to (Reference 1) is employed. DNA sequence mutation to achieve the desired amino acid substitution can be performed by using the overlap extension method or cassette mutation method that uses the cloned DNA before modification as a template and a synthetic oligo DNA incorporating mismatched base pairs as a primer for polymerase chain reaction. Etc. can be easily introduced into the intended site.
Furthermore, when protein A-derived immunoglobulin binding protein is used as a ligand for immunoglobulin affinity chromatography, a multimeric protein in which 2 or more, preferably about 4 immunoglobulin binding domains are linked is produced. Is being used. The immunoglobulin-binding protein obtained by the present invention is also preferably produced and used as a multimeric protein in which two or more immunoglobulin-binding domains are linked and desirably about four. A cDNA encoding such a multimeric protein can be easily prepared by connecting a desired number of cDNAs encoding an immunoglobulin binding domain in series. By inserting the cDNA thus prepared into an appropriate expression plasmid and using it, it is possible to easily produce a multimeric protein in which two or more immunoglobulin binding domain units are linked.

  As an expression vector into which the nucleic acid sequence encoding the modified protein of the present invention is inserted, any vector such as a plasmid, a phage, or a virus that can replicate in a host cell can be used. For example, commercially available expression vectors include pQE vectors (Qiagen), pDR540, pRIT2T (GE Healthcare Biosciences), pET vectors (Merck). The expression vector should be used by selecting an appropriate combination with the host cell. For example, when Escherichia coli is used as a host cell, a combination of a pET vector and a BL21 (DE3) E. coli strain or a combination of a pDR540 vector and a JM109 E. coli strain is preferable.

  The modified protein of the present invention can be recovered in a soluble fraction by collecting cultured cells by centrifugation or the like and crushing them by treatment using ultrasonic waves, a French press or the like. Purification of the modified protein can be performed by appropriately combining known separation and purification techniques. Specifically, in addition to separation techniques such as salting out, dialysis, and ultrafiltration, purification methods such as hydrophobic chromatography, gel filtration chromatography, ion exchange chromatography, affinity chromatography, and reverse phase chromatography are exemplified. The modified protein of the present invention has been modified to reduce the negative charge on the surface and increase the positive charge, and has a characteristic that the isoelectric point is higher than many contaminating proteins derived from host cells such as E. coli. It is particularly desirable to employ ion exchange chromatography in the purification process.

  Examples of the insoluble carrier to which the modified protein of the present invention is bound as an immunoglobulin affinity ligand include natural polymer materials such as chitosan and dextran, and synthetic polymers such as vinyl polymers, highly cross-linked agarose, and polyimide. . In another form, an inorganic carrier such as silica may be used. Usually, the ligand protein is supported by a coupling agent such as cyanogen bromide, epichlorohydrin, N-hydroxysuccinimide, tosyl / tresyl chloride, carbodiimide, glutaraldehyde, hydrazine or a carboxyl or thiol activated carrier. Fixed on top. Such coupling reactions are well known in the art and are widely described in the literature (eg, Jansson, JC and Ryden, L., “Protein purification”, 2nd edition, pages 375-442, ISBN). 0-471-18626-0). The ligand protein of the present invention is characterized in that it is bound to a carrier via a plurality of amino groups arranged so that the orientation of the ligand can be controlled spatially. For immobilization of the protein, tresyl is used. A carrier having an active group capable of forming a covalent bond by reacting with a protein amino group such as a group, an epoxy group, a carboxyl group, or a formyl group can be used. Commercially available carriers include Toyopearl AF-Tresyl-650, Toyopearl AF-Epoxy-650, Toyopearl AF-carboxy-650, Toyopearl AF-Formyl-650 (above, Tosoh Corporation), NHS-activated Sepharose, cyanogen bromide activity Sepharose, epoxy-activated sepharose (GE Healthcare Bioscience Co., Ltd.) and the like.

  The thus produced affinity carrier of the present invention can be used for affinity chromatography in the process of isolating and purifying immunoglobulins such as IgA, IgG, IgM, etc. by packing them in an appropriate column.

  In another embodiment, the immunoglobulin-binding protein of the present invention can also be used for the purpose of binding immunoglobulin in a form immobilized on a substrate such as a capillary, chip or filter. As for the immobilization reaction to the base material at this time, it is desirable to employ a reaction with an active group capable of forming a covalent bond by reacting with the amino group of the protein as in the immobilization onto the carrier for affinity chromatography.

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.
First, for the modified protein expressed as a His-tag added protein, the convenience of the purification operation was evaluated from the behavior in cation exchange chromatography, and the stability under acidic pH conditions was evaluated using the purified modified protein. Next, a modified protein not added with His-tag was prepared and purified and immobilized on a chromatographic support. The resulting affinity chromatography gel was used to evaluate immunoglobulin binding activity and stability under alkaline pH conditions.

[Construction of expression plasmid for His-tag added immunoglobulin binding protein]
First, two pairs of double-stranded DNA fragments obtained from four synthetic oligonucleotides were joined together, and the DNA fragment of the sequence shown in FIG. 1 (SEQ ID NO: 4 in the sequence listing) was ligated with pUC19 plasmid (stock) using EcoRI and HindIII. PUC / ASR1 was obtained by cloning onto Nippon Gene).
Next, three pairs of double-stranded DNA fragments obtained from six synthetic oligonucleotides between the restriction enzyme cleavage sites of EcoRI-AflII on this plasmid were converted into four synthetic oligos between the restriction enzyme cleavage sites of AflII-NheI. Two pairs of double-stranded DNA fragments obtained from nucleotides were sequentially joined and cloned, and the immunoglobulin binding protein shown in FIG. 2 (protein in which glycine at position 29 of the C domain was substituted with alanine (SEQ ID NO: 1 in the Sequence Listing) PUC / SPAC ′, which contains a DNA sequence (SEQ ID NO: 5 and 6 in the sequence listing) corresponding to SPAC ′).
Next, a cDNA fragment encoding a protein was excised from this plasmid using NdeI and BamHI, and incorporated into a pET-9a plasmid (Merck Co., Ltd.) to obtain an SPAC 'expression plasmid, pET / SPAC'. (Figure 3).
Next, in order to construct a plasmid encoding a mutant in which one amino acid was substituted, PCR was performed using the synthetic oligonucleotide pairs shown in Table 1 as primers and the pUC / SPAC 'plasmid as a template. A cDNA fragment containing the mutation was prepared. PrimeSTAR HS DNA polymerase (Takara Bio Inc.) was used as the PCR enzyme, and PCR Thermal Cycler SP (Takara Bio Inc.) was used as the apparatus.
These fragments were then recombined on the pUC / SPAC 'plasmid using the restriction enzymes shown in Table 1, respectively, and V40K, E43K, A46K, D53K, A54K, A56K, and three lysines were inserted at the C-terminus. A plasmid of -61K mutant was prepared.
By using the thus obtained amino acid substitution mutant plasmid as a template, PCR was performed to prepare mutant cDNA fragments containing amino acid substitutions at a plurality of sites, and these fragments were respectively used with the restriction enzymes shown in Table 1. Then, recombination was carried out on a plasmid having a mutation at another site, and a plasmid in which two or more amino acids were substituted was constructed.
Table 1 shows combinations of PCR primers used for construction of amino acid substitution displacement plasmids, plasmids used as templates and subcloning vectors, and restriction enzymes used for recombination of DNA fragments. The sequence number of each primer represents the sequence number in the sequence listing.
The nucleic acid sequence of the portion encoding the recombinant protein in each expression plasmid was confirmed using a CEQ8000 type DNA sequencer (Beckman Coulter, Inc.).

[Examination of expression and purification process of His-tag added immunoglobulin binding protein]
BL21 (DE3) competent cells (Merck Co., Ltd.) were transformed with each of the constructed expression plasmids to obtain strains expressing the modified proteins.
Each E. coli strain was inoculated into 2 × TY medium containing 25 mg / L kanamycin and cultured at 37 ° C. for 16 hours to express the target protein, and then E. coli was collected by centrifugation.
Next, the collected E. coli was suspended in 50 mM MES buffer (pH 6.0), sonicated to disrupt the E. coli, and the target protein was recovered in the supernatant by centrifugation.
After filtering this sample solution using ULTRAFREE MC 0.45 μm Filter Unit (Nippon Millipore Corporation), it was applied to an SP-5PW column (7.5 × 75 mm; Tosoh Corporation), and 50 mM MES buffer (pH 6). 0.0) as the mobile phase, and the protein of interest was eluted with a linear concentration gradient of sodium chloride at a flow rate of 1 mL / min.
FIG. 4 shows the behavior of the modified protein in which the immunoglobulin binding protein lysine is increased by 3 to 5 in cation exchange chromatography. Increased lysine residues and increased cation charge increased the binding power of immunoglobulin-binding proteins to the cation exchange column, confirming that they elute at higher salt concentrations. As a result, modification of lysine introduction It was found that the body can be easily separated from contaminating proteins derived from E. coli.
The immunoglobulin-binding protein used in this study has His-tag added to the C-terminus, but SPAC ', which does not have His-tag, has a pKa of 1 compared to the protein to which His-tag is added. Since it is low to the extent, when purifying a modified protein having no His-tag used as an immunoglobulin affinity ligand, the effect of increasing the positive charge by the modification of the present invention becomes larger. That is, FIG. 4 shows that the immunoglobulin-affinity ligand of the present invention can dramatically simplify the production process and reduce the cost as compared with the conventional product.

[Study on stability of modified immunoglobulin-binding protein excluding aspartate-proline sequence under acidic pH conditions]
SPAC ′ purified according to Example 2 and a modified immunoglobulin binding protein in which the aspartic acid at position 37 was replaced with other amino acids at a protein concentration of 0.5 mg / mL in water or 50% formic acid at 37 ° C. Keep warm for hours.
The solution after the treatment was diluted 50 times with 50% acetonitrile containing 0.1% trifluoroacetic acid, 1 μL of the diluted solution was spotted on an NP20 chip, and analyzed by SELDI-TOF MS (Bio-Rad Laboratories, Inc.).
As shown in FIG. 5, in the sample of SPAC ′ treated under acidic pH conditions, peptide fragments generated by cleavage between aspartic acid and proline at positions 37-38 were detected by mass spectrometry. No cleaved fragments were detected from the treated variant samples. That is, it is shown that the immunoglobulin binding protein before modification is susceptible to cleavage between aspartic acid and proline at positions 37-38 under acidic pH conditions, and this defect is eliminated by the amino acid substitution at position 37 of the present invention. It was.

[Construction of expression plasmid for immunoglobulin-binding protein without adding His-tag]
First, a primer without a His-tag coding sequence was designed at the C-terminal using the His-tag-added modified immunoglobulin-binding protein expression plasmid as a template, and His-tag was encoded by PCR using this primer. A desired DNA fragment having no sequence to obtain was obtained.
This DNA fragment was cleaved with NdeI and BamHI, and then incorporated into the pET-9a plasmid to prepare a modified immunoglobulin-binding protein expression plasmid without His-tag.
Next, amino acids at positions 4, 7, and 35 by the overlap extension method (two-step PCR method) using the modified expression plasmid having no sequence encoding His-tag as a template and the primers shown in Table 2 A mutation causing substitution was introduced and similarly incorporated into the pET-9a plasmid to prepare the desired expression plasmid. Further, a mutant cDNA fragment containing amino acid substitutions at a plurality of sites was prepared by PCR using the obtained mutant plasmid as a template and a primer that causes mutation at another position. Further, a modified expression plasmid in which amino acids at 2 to 10 sites were substituted was constructed by an overlap extension method combining cDNA fragments having mutations introduced into the portion before and after position 35.
The sequence number of each primer represents the sequence number in the sequence listing.

[Expression and purification of immunoglobulin-binding protein without adding His-tag]
After confirming that the nucleic acid sequence of the expression plasmid of the immunoglobulin-binding protein without the added His-tag was as intended, each plasmid was transformed into BL21 (DE3) competent cell (Merck Co., Ltd.), The same culture as when the His-tag-added immunoglobulin binding protein was expressed was performed to obtain Escherichia coli expressing the target protein.
The obtained Escherichia coli was suspended in 20 mM phosphate buffer (pH 6.0), sonicated to disrupt the Escherichia coli, and the target protein was recovered in the supernatant by centrifugation.
0.2M acetic acid is added to this sample solution to adjust the pH of the solution to about 0.6 lower than the theoretical isoelectric point of the modified protein, and then centrifuged again to recover the target protein in the supernatant. The solution was filtered using ULTRAFREE MC 0.45 μm Filter Unit (Nippon Millipore Corporation), then applied to a Toyopearl Gel SP-550C packed column (5 mL; Tosoh Corporation), and 50 mM sodium acetate buffer (pH 4. 5) or 50 mM MES buffer (pH 6.0) was used as a mobile phase to purify the target protein by eluting with a linear concentration gradient of sodium chloride.
The purity of the purified protein was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the protein concentration was determined using BCA Protein Assay (Pierce Biotechnology Co., Ltd.).
By this operation, it was possible to obtain about 200 mg of purified product per liter of the culture solution for any modified immunoglobulin binding protein.

[Immobilization of a modified immunoglobulin-binding protein without adding a His-tag to a chromatography carrier]
The purified immunoglobulin-affinity ligand solution of the present invention is subjected to gel filtration using Sephadex G-25 gel (GE Healthcare Bioscience Co., Ltd.), so that a buffer solution (0. It was replaced with 0.2M sodium hydrogen carbonate buffer (pH 8.3) containing 5M NaCl.
Ligand immobilization reaction was performed by adding an equal volume of the ligand solution adjusted to a concentration of 2 mg / mL to the gel after washing with NHS-activated Sepharose 4-FF gel (GE Healthcare Biosciences) at 25 ° C. Shake for 4 hours.
After completion of the immobilization reaction, the supernatant of the reaction solution was collected, applied to a TSK gel SuperSW column (4.6 × 300 mm; Tosoh Corporation), and 0.5 mM NaCl-containing 50 mM phosphate buffer (pH 7. In gel filtration chromatography using 0) as the mobile phase, unreacted ligand protein was detected at an absorbance of 280 nm, the amount of unreacted ligand was measured, and the amount of ligand immobilized on the gel was determined.
As a result, for all the immunoglobulin affinity ligands of the present invention, it was confirmed that 99% or more of the amount used for the immobilization reaction under the present conditions was immobilized on the gel.
The gel after the ligand immobilization reaction blocks the unreacted NHS activating group by shaking in a 0.5M ethanolamine solution (pH 8.3) containing 0.5M NaCl for 1 hour at 25 ° C. And then washed three times alternately with 0.1 M sodium acetate solution (pH 4.0) containing 0.5 M NaCl and 0.5 M ethanolamine solution (pH 8.3) containing 0.5 M NaCl, and finally phosphoric acid. After washing 3 times with buffered saline (hereinafter referred to as PBS), it was used to measure the amount of immunoglobulin bound.

[Measurement of immunoglobulin binding amount of affinity gel using modified immunoglobulin binding protein as ligand]
An equal volume of 40 mg / mL human immunoglobulin (Sigma) in PBS was added to the ligand-immobilized gel, and the mixture was shaken at 25 ° C. for 1 hour to adsorb the immunoglobulin to the gel. Next, the gel was washed three times with PBS, and then the immunoglobulin was eluted from the gel by shaking in 0.1 M glycine-hydrochloric acid buffer (pH 2.8) at 25 ° C. for 10 minutes.
The eluate was neutralized by adding 1/20 volume of 1M Tris solution, the absorbance at 280 nm was measured, and the immunosorbent adsorbed on the affinity gel based on the specific absorption coefficient of 13.8 (1 g-1 cm-1). The amount of globulin was determined.
When the immunoglobulin binding protein according to the present invention is used as an affinity ligand with the value (IgG / ligand ratio) obtained by dividing the amount of adsorbed immunoglobulin (mg) per 1 ml of gel by the amount of immobilized ligand (mg) as an index. The results of evaluating the immunoglobulin binding activity are shown in FIG.
At present, the best mode of the present invention for immunoglobulin binding activity is to substitute lysines at positions 4, 7 and 35 of the immunoglobulin binding domain of Staphylococcus protein A with amino acids other than lysine, and Modification of substitution of 3 to 5 amino acids among amino acids at positions 40, 43, 46, 53 and 56 on the protein surface, and addition of 3 lysines to the C-terminal The affinity gel using these modified proteins as affinity ligands showed an immunoglobulin binding activity up to 3.5 times that of the modified protein using the SPAC ′ domain before modification.
Among the amino acid sequence modifications encompassed by the present invention, the modification in which lysine at position 35 is substituted with an amino acid other than lysine increases the amount of immunoglobulin adsorbed on the gel immobilized with the obtained protein as an affinity ligand. The effect was extremely high, and substitution with arginine or glutamine was most effective. This result shows that when the immunoglobulin A binding domain of protein A is immobilized on a gel in the vicinity of position 35, the molecule has a markedly reduced activity of binding immunoglobulins. It should be noted that an immunoglobulin-binding protein (described in Patent Document 1), which is another protein derived from Staphylococcus and different from protein A, has about 45% homology in the amino acid sequence with the immunoglobulin-binding domain of protein A. A region having a arginine is present at a position corresponding to position 35 of this sequence. However, at position 1-38, which is presumed to be an immunoglobulin-binding region in this similar domain, there are three lysines that are completely different from the immunoglobulin-binding domain of protein A, in particular at position 32, in addition to position 39. The number of lysines present thereafter is small compared to the immunoglobulin binding domain of protein A, and the ratio of the number of lysine after position 39 to the number of lysine at position 1-38 is even lower than that of the immunoglobulin binding domain of protein A. Therefore, it is easily guessed that it is extremely difficult to maintain good orientation when immobilizing on a carrier through a lysine residue. That is, it is clear that the modified protein of the present invention has structural and functional characteristics that are remarkably useful as an affinity ligand for immunoglobulin as compared with the protein described in Patent Document 1.

[Study of the stability of affinity gel with modified immunoglobulin-binding protein as a ligand under alkaline pH conditions]
An affinity gel using the modified immunoglobulin-binding protein as a ligand was kept warm in a 0.1N NaOH solution at 25 ° C. for a fixed time, washed three times with PBS, and the amount of immunoglobulin binding was measured according to the method of Example 7.
The amount of immunoglobulin binding after treatment under alkaline pH conditions was measured at each treatment time, and the residual ratio when the amount of immunoglobulin binding before treatment under alkaline pH conditions was taken as 100% is shown in FIG.
This result shows that the immunoglobulin affinity ligand of the present invention in which the lysine residue on the protein surface of helix 3 is increased in the immunoglobulin binding domain of Staphylococcus protein A is immobilized in an affinity gel, and is alkaline. It is more stable than the unmodified ligand for treatment under pH conditions.

  The carrier for affinity chromatography using the immunoglobulin binding protein according to the present invention is inexpensive, has a high ability to bind immunoglobulin, and has little leakage of ligand protein. That is, the present invention is useful for the production of inexpensive and high-quality immunoglobulins, the construction of antibody-immobilized chips and microplates used for diagnosis, and highly safe blood purification systems for removing immunoglobulins.

It is drawing which shows the nucleic acid sequence of the synthetic oligonucleotide used for construction of pUCASR1 plasmid. FIG. 2 shows nucleic acid and amino acid sequences encoding immunoglobulin binding protein constructed on pUC / SPAC 'plasmid. 1 is a diagram showing a procedure for constructing an expression plasmid for an immunoglobulin binding protein. It is the figure which showed the behavior in the cation exchange chromatography of the immunoglobulin binding protein variant. It is drawing which compared the to-be-cut | disconnected characteristic when processing immunoglobulin binding protein on acidic pH conditions. It is drawing which compared the IgG binding ability of immunoglobulin binding protein when it fix | immobilizes as an affinity ligand to the support | carrier for chromatography. It is the figure which compared the residual activity when processing the affinity gel which uses immunoglobulin binding protein as a ligand on alkaline pH conditions.

Claims (13)

An immunoglobulin binding protein capable of binding to a region other than the complementarity determining region (CDR) of an immunoglobulin molecule, wherein the variant is a modified C domain of Staphylococcus protein A defined by SEQ ID NO: 1 or Modification that substitutes lysine from any one of positions 4, 7, and 35 in the Z domain defined by SEQ ID NO: 2 with an amino acid other than lysine (however, only lysine originally from position 35 is converted to alanine (Excluding modification to be substituted)), when the protein is immobilized on an insoluble carrier via its own amino group, the orientation for maintaining affinity for immunoglobulin is improved compared to the molecule before modification. A modified protein characterized by   The modified protein according to claim 1, wherein in addition to the modification, 1 to 6 of amino acids at positions 40, 43, 46, 53, 54, and 56 are substituted with lysine. .   The modification of substituting lysine from any one or more of positions 4, 7, and 35 with an amino acid other than lysine replaces lysine from any one or more of positions 4, 7, or 35 with alanine, glutamine, or asparagine. , Valine, serine, threonine, histidine, tyrosine, or arginine (except for the modification that replaces only the original lysine at position 35 with alanine). protein. In addition to the above modification, further, 3 to 37-position of aspartic acid by modification of substituting an amino acid other than aspartic acid, claim 1, chemical stability at acidic pH conditions are improved as compared to a molecule of the unmodified The modified protein according to any one of the above. 5. The modification according to claim 4 , wherein the modification of substituting aspartic acid at position 37 with an amino acid other than aspartic acid is that the amino acid at position 37 is substituted with alanine, glutamic acid, serine, threonine, leucine, or isoleucine. Modified protein.   The modified protein according to claim 1, comprising the amino acid sequence defined by SEQ ID NO: 3. A multimer characterized in that 2 to 5 immunoglobulin-binding protein units are linked, and the modified protein according to any one of claims 1 to 6 is contained therein. A nucleic acid encoding the modified protein according to any one of claims 1 to 7 , or a multimer containing them. A gene expression system comprising the nucleic acid according to claim 8 . A carrier for affinity chromatography comprising the modified protein according to any one of claims 1 to 7 or a multimer containing them as an affinity ligand. An affinity column comprising the affinity chromatography carrier according to claim 10 . A method for affinity separation of IgG, IgA and / or IgM, wherein the affinity column according to claim 11 is used. A protein chip comprising the modified protein according to any one of claims 1 to 7 , or a multimer containing them.