WO2015093507A1 - Novel modified protein of extracellular domain of protein g - Google Patents

Abstract

The purpose of the present invention is to provide a novel protein, or the like, which has reduced bindability with the Fc region and/or the Fab region of an immunoglobulin in a weakly acidic range and has an elution pH which has been shifted towards weak acidity. The present invention pertains to: a protein that has a binding activity to the Fc region of immunoglobulin G and, as a result of substituting a residue in the vicinity of a positively charged residue of protein G for the positively charged residue, has a reduced binding activity to the Fc region of immunoglobulin G in a weakly acidic range compared to a protein comprising a wild-type protein G B domain; a scavenger of immunoglobulin G or of a protein having the Fc region or the Fab region of immunoglobulin G, wherein the protein is immobilised on a water-insoluble solid-phase support; affinity chromatography for purification of immunoglobulin G or of a protein having the Fc region or the Fab region of immunoglobulin G, said affinity chromatography including the scavenger; and a method which uses the affinity chromatography for purification and in which immunoglobulin G or a protein having the Fc region or the Fab region of immunoglobulin G is purified.

Description

Novel modified protein of extracellular domain of protein G

The present invention is a protein comprising a conventional wild type protein G / B domain (extracellular domain), or a variant of the protein, wherein the binding activity in the weakly acidic region to the Fc region of immunoglobulin G is reduced. In addition to the improved protein (domain variant), the binding activity in the weakly acidic region to the protein having the Fc / Fab region of immunoglobulin G (hereinafter referred to as “pH-responsive variant”) is also reduced. For example).

Conventionally, purification of proteins such as antibodies has been an important issue in biochemical research, and various techniques such as affinity chromatography, gel filtration chromatography, and ion exchange chromatography are known. Affinity chromatography is a method for purifying a target protein using specific affinity with the target protein. In this method, proteins can be easily and selectively recovered. However, since the affinity is very strong, in order to dissociate the protein adsorbed on the chromatographic packing material, the pH is generally about 2.5. Elution with acidic buffer is often required. Under such strongly acidic conditions, activity reduction such as protein denaturation is likely to occur, and purification under milder conditions is required.

Protein G is a membrane protein present in the cell membrane of Streptococcus spp. And is known to have specific binding activity to the Fc region of immunoglobulin G, which is a kind of antibody (Non-patent Document 1, Patent Document 1). ). Protein G is a multidomain membrane protein composed of a plurality of domains, and shows a binding activity to a protein having an Fc region of immunoglobulin G (hereinafter referred to as “antibody binding activity”). It is an extracellular domain (Non-patent Document 2). For example, in the case of protein G derived from the G148 strain shown in FIG. 1 of Patent Document 8, three domains B1, B2, and B3 exhibit antibody binding activity (also referred to as C1, C2, and C3 domains in the literature). ) In addition, there are three antibody binding domains in protein G of GX7805 strain and two antibody binding domains in protein G of GX7809. These are all small proteins of less than 60 amino acids and are known to show high identity between their amino acid sequences). In addition, it is known that antibody binding activity is maintained even when protein G is cleaved to isolate each domain alone (Non-patent Document 3).

As for the extracellular domain of protein G, many products containing protein G extracellular domain utilizing its selective antibody binding activity are currently on the market (for example, carriers for affinity chromatography for antibody purification (patent documents) 3, 4), test reagents for detecting antibodies, research reagents, etc.). It is known that the binding force between an extracellular domain of protein G and an antibody is high in a neutral to weakly acidic region and low in a strongly acidic region (Non-patent Document 4). Therefore, for the purpose of antibody isolation, recovery, and purification, first, a sample solution containing an antibody such as serum is neutralized, and a water-insoluble solid phase such as beads immobilized with the extracellular domain of protein G is immobilized. The antibody is selectively adsorbed by contacting with a phase support. Thereafter, washing with a neutral to weak acid solution (pH 5 to 8) is performed to remove components other than antibodies. Finally, it is common to add a strongly acidic solution of pH 2.4 to 3.5 to desorb the antibody from the immobilized protein G and to elute it together with the strongly acidic solution (Patent Document 3). Thereby, the antibody can be isolated, recovered and purified with high purity.

However, when an antibody is placed in a strongly acidic solution having a pH of 2.4 to 3.5, it may deteriorate due to denaturation aggregation or the like, and depending on the type of antibody, the original function may be lost (Non-patent Document 4). In order to prevent this, treatment in a weakly acidic region at a pH higher than 2.4-3.5 is attempted, but since the binding force between the extracellular domain of protein G and the antibody is strong, in the weakly acidic region the antibody is separated from protein G. It does not elute and a sufficient recovery amount cannot be obtained. On the other hand, protein G extracellular domain is known to bind to Fab (Non-patent Document 2), and one antibody molecule can bind to protein G extracellular domain in two regions, Fc region and Fab region. is there. In such a binding state, the antibody and the extracellular domain of protein G cannot be easily dissociated, making it difficult to recover the antibody.

To date, the present inventors have described a protein G having thermal stability, chemical stability against denaturing agents, resistance to proteolytic enzymes, etc. (these properties are collectively referred to simply as “protein stability”). Improved protein comprising an extracellular domain mutant of the above (Patent Document 5 and Patent Document 6), and further, has a binding property to an immunoglobulin Fc region and / or a binding property to the Fab region in a weakly acidic region. A reduced improved protein was also developed (Patent Document 7). However, all of these improved proteins contain only one domain exhibiting antibody binding activity.

Furthermore, the present inventors have developed a protein comprising a tandem multimer of the improved protein (Patent Document 8). Compared with the tandem multimer of the wild-type protein G / B1 domain, such a protein has a much lower binding property in the weakly acidic region with the Fc region of human immunoglobulin G of different subclasses IgG1 and IgG3. In the chromatographic column for protein separation and purification packed with the capture agent of the present invention containing the protein, the captured human immunoglobulin G can be more easily obtained in a weakly acidic region (about pH 4 to 5) without denaturation. It became possible to elute.

However, in order to avoid denaturation of some antibodies, there is still a demand for a method for purifying antibodies that can be eluted at a slightly acidic pH.

Japanese National Publication No. 03-501801 Japanese Patent No.2764021 Japanese Unexamined Patent Publication No. 03-128400 JP 2003-088381 A JP 2009-95322 JP JP 2009-118749 JP 2009-297018 International Publication No.2013 / 018880

Thus, the problem to be solved by the present invention is that the binding to the Fc region of immunoglobulin and / or the Fab in the weakly acidic region further compared with the conventional wild-type protein G / B domain or its modified protein. It is to provide a novel protein having a reduced binding property to a region and a more acidic pH shifted to a slightly acidic side.
Furthermore, the problem to be solved by the present invention is to provide a chromatography column for protein separation and purification, particularly an affinity chromatography column for antibody purification, which is packed with the capture agent.

In the conventional wild-type protein G · B domain or a domain variant thereof, the present inventor has a positive charge that exists within a certain distance from the Fc region in the domain variant / Fc complex model structure of wild-type protein G. We found that by substituting amino acid residues near the residues with positively charged residues, the binding activity of these proteins to the Fc region of immunoglobulin G in the weakly acidic region can be significantly reduced. Completed the invention.

That is, each aspect of the present invention is as follows.
[Aspect 1]
Compared to the protein consisting of wild-type protein G and B domains, it has binding activity to the Fc region of immunoglobulin G. By substituting residues near the positively charged residues of protein G with positively charged residues A protein with reduced binding activity in the weakly acidic region to the Fc region of immunoglobulin G.
[Aspect 2]
The protein according to embodiment 1, wherein the amino acid residue after mutation is histidine.
[Aspect 3]
Neighboring residues are within 6 angstroms from any amino acid residue in the Fc region in the wild type protein G / Fc complex crystal structure or the domain variant / Fc complex model structure of wild type protein G derived therefrom The protein according to embodiment 1 or 2, which is an amino acid residue within 6 angstroms from a positively charged residue of wild-type protein G or a domain variant thereof.
[Aspect 4]
The protein according to any one of aspects 1 to 3, wherein the wild-type protein G · B domain is any one of B1, B2 and B3 of protein G of Streptococcus spp.
[Aspect 5]
The protein according to any one of aspects 1 to 4, wherein the wild-type protein G · B domain has an amino acid sequence represented by SEQ ID NO: 30, and the domain variant thereof is represented by any one of SEQ ID NOs: 31 to 38.
[Aspect 6]
The amino acid residue at positions 25, 36 and 41 in the sequence of the protein G / B domain or a domain variant thereof is mutated to histidine according to any one of embodiments 1 to 5. protein.
[Aspect 7]
The protein according to any one of embodiments 1 to 6, wherein the antibody dissociation rate in the pH4 solution is higher than that of wild-type protein G · B or a domain variant thereof as measured by the SPR method.
[Aspect 8]
A protein which is a tandem multimer of the protein according to any one of aspects 1 to 7.
[Aspect 9]
The protein according to embodiment 6, which is a trimer, tetramer, or pentamer.
[Aspect 10]
The protein according to embodiment 8 or 9, wherein the extracellular domain mutants constituting the multimer are identical to each other.
[Aspect 11]
The protein according to any one of embodiments 8 to 10, wherein each extracellular domain variant is linked by a linker sequence.
[Aspect 12]
A protein which is a fusion protein comprising an amino acid sequence obtained by linking the amino acid sequence of the protein according to any one of embodiments 1 to 11 and the amino acid sequence of another protein.
[Aspect 13]
The protein according to embodiment 12, which has the amino acid sequence represented by any of SEQ ID NO: 21, 23 or 24.
[Aspect 14]
A nucleic acid encoding the protein according to any one of Embodiments 1 to 13.
[Aspect 15]
A recombinant vector containing the nucleic acid according to Aspect 14.
[Aspect 16]
A transformant into which the recombinant vector according to aspect 15 has been introduced.
[Aspect 17]
An immunoglobulin G, or a protein having an Fc region or Fab region of immunoglobulin G, wherein the protein according to any one of embodiments 1 to 13 is immobilized on a water-insoluble solid phase support. Scavenger.
[Aspect 18]
Affinity chromatography for purification of immunoglobulin G or a protein having an Fc region or Fab region of immunoglobulin G, comprising the capture agent according to aspect 17.
[Aspect 19]
A method for purifying an immunoglobulin G or a protein having an Fc region or Fab region of immunoglobulin G using the affinity chromatography for purification according to aspect 18.

According to the present invention, there is a residue near the positively charged residue of protein G in an acidic solution as compared with a protein having a binding activity to the Fc region of immunoglobulin G and consisting of wild-type protein G and B domains. By substituting with a positively charged residue, it is possible to provide a protein having reduced binding activity in the weakly acidic region to the Fc region of immunoglobulin G.

As a result, in the chromatography column for protein separation and purification packed with the capture agent of the present invention containing the protein, the captured antibody such as immunoglobulin G is more easily eluted in a weakly acidic region without denaturation. It becomes possible.

It is a schematic diagram of a site-specific mutagenesis PCR reaction and a template plasmid cleavage reaction. It is a SPR measurement result of improved protein G (PG19). After adding the antibody sample, each pH solution (1st cycle: pH 5, 2 cycle: pH 4, 3 cycle: pH 3, 4 cycle: pH 2) was added. It is a SPR measurement result of the histidine additional mutant (T25H, E36H, G41H, T55H) belonging to the group near the binding interface. After adding the antibody sample, each pH solution (1st cycle: pH 5, 2 cycle: pH 4, 3 cycle: pH 3, 4 cycle: pH 2) was added. It is a SPR measurement result of a histidine additional mutant (D1H, N8H, E15H, Y33H, T56H) belonging to a group away from the binding interface. After adding the antibody sample, each pH solution (1st cycle: pH 5, 2 cycle: pH 4, 3 cycle: pH 3, 4 cycle: pH 2) was added. It is a graph shown regarding the calculation criteria etc. of the dissociation rate (%) and leak (%) from a sensorgram. It is a graph which shows the result of pH gradient affinity chromatography using a recombinant PG fixed column. The outline of the design concept of the structure of the protein of the present invention is shown.

Protein G, a streptococcal protein, is known to have a specific binding activity to the Fc region of immunoglobulin G, which is a kind of antibody (Reference Document 1). It is a protein useful for purification, removal, diagnosis, treatment, testing, etc. using antibodies. Protein G is a multidomain membrane protein composed of a plurality of domains, and shows a binding activity to a protein having an Fc region of immunoglobulin G (hereinafter referred to as “antibody binding activity”). It is an extracellular domain (Reference Document 2). For example, in the case of protein G derived from the G148 strain, three domains B1, B2, and B3 exhibit antibody binding activity (also referred to as C1, C2, and C3 domains in the literature). In addition, there are three antibody binding domains in protein G of GX7805 strain, and two antibody binding domains in protein G of GX7809. These are all small proteins of less than 60 amino acids, and high identity is seen between their amino acid sequences. In addition, it is known that antibody binding activity is maintained even if protein G is cleaved to isolate each domain alone (Reference 3).

The present invention has a binding activity to the Fc region of immunoglobulin G, and a residue near the positively charged residue of protein G as a positively charged residue compared to a protein consisting of wild type protein G and B domains. It relates to a protein whose binding activity in the weakly acidic region to the Fc region of immunoglobulin G is reduced by substitution.

Here, examples of immunoglobulin G include human and non-human animals, particularly various antibodies of mammals such as rats, mice, hamsters, goats and rabbits, and various fragments of antibodies such as Fab fragments of human IgG. including. As long as it has an Fc part or Fab part, its structure or component is not particularly limited, and includes any of various types of antibody molecules and their fragment molecules known to those skilled in the art. That is, in addition to normal (complete) IgG type antibody molecules, for example, single chain antibodies (scFv), single chain antibody dimers, bispecific antibodies, diabody type bispecific antibodies, And multimerized low molecular weight antibodies, and various antibody fragments such as Fab fragments, F (ab ′) ₂ and Fab ′.

In the protein of the present invention, “the binding activity in the weakly acidic region with respect to the Fc region of immunoglobulin G is reduced” means that, for example, as shown in the Examples of the present specification, pH 4 is measured in the SPR method. This means that the antibody dissociation rate (%) in the solution is higher than that of the wild type protein G · B or its domain variant, preferably about 2 to 30 times higher than that of the domain variant. Alternatively, the column in which the protein of the present invention is immobilized has an elution peak of immunoglobulin G such as human IgG in pH gradient affinity chromatography as compared to a column in which wild type protein G • B or a domain variant thereof is immobilized. This means that it shifts to the neutral side (pH value is about 0.6 to 2.6).

Substituting a residue in the vicinity of a positively charged residue of protein G in the protein of the present invention with a positively charged residue specifically includes one to several positively charged residues of protein G (in acidic solution) For example, in the hope that an electrostatic repulsion will occur between a lysine residue or a histidine residue) and its nearest residue, the closest residue of the positively charged residue of protein G is changed to a positively charged residue. To replace.

Here, the distance from the positively charged amino acid residue substituted with the positively charged residue in the wild type protein G • B or its domain variant is the origin of the charged atom of the positively charged residue, and any substituted amino acid With the heavy atom in the side chain of the residue as the end point, the smallest value among these distances is defined as the distance between the residues.

Here, the type of amino acid of the mutated residue is not particularly limited, but the mutated amino acid residue is preferably histidine.

The positively charged residue of wild type protein G or its domain variant is the same as the Fc region in the wild type protein G / Fc complex crystal structure or the wild type protein G domain variant / Fc complex model structure derived therefrom. From the viewpoint of the pH responsiveness of the protein of the present invention, it is preferably in the vicinity of the binding interface, for example, within 6 angstroms from any amino acid residue in the Fc region.

Further, the distance between the positively charged residue and the mutated positively charged amino acid residue is preferably within about 6 angstroms in the above crystal structure or model structure.

The design concept (design procedure) of the structure of the protein of the present invention having such characteristics is as follows.
[Example of design procedure using PG19 described in Patent Document 8 (see FIG. 7)]
(1) Based on the wild-type PG / Fc region complex crystal structure (pdb, 1FCC) and the PG19 crystal structure (pdb, 2ZW1), the PG19 / Fc region complex structure is modeled. Complex modeling is performed by software superpose (non-patent literature @@ E. Krissinel and K. Henrick (2004) Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Cryst. D60, 2256-2268) is used for molecular structure calculation by software MOE (Ryoka System).
(2) The distance between atoms within the PG19 / Fc region molecule and within the PG19 molecule is calculated using CONTACT included in the software package CCP4 (Non-patent Document 6).
(3) Based on the above definition, the closest residue in PG19 from the positively charged residue (☆) included in PG19 is the residue to be mutated (◯).
(4) Of the residues to be mutated (O), based on the binding analysis of the alanine substitution performed in the previous research (Non-patent Document 7), it became clear that it is important for the binding of PG19 and Fc region. Exclude those that are. If the free energy difference DDG is positive, it indicates that it is disadvantageous for coupling.
(5) Based on the above definition, the distance from the positively charged residue (☆) contained in PG19 to the closest Fc region residue (□) is determined.

Information on the atoms selected for calculating the inter-atomic distance between the PG19 / Fc region molecules and the PG19 molecule using the software CONTACT is as follows.
For positively charged residues (☆) contained in PG19, select a charged nitrogen atom. For the residue in PG19 closest to the positively charged residue (☆), the closest side chain atom (main chain atom in the case of glycine) is selected from the above positively charged nitrogen atom. For the residue in the Fc region closest to the positively charged residue (☆), the closest side chain atom (main chain atom in the case of glycine) is selected from the above positively charged nitrogen atom.

Examples of the wild-type protein G · B domain include any of B1, B2 and B3 of protein G of Streptococcus genus Streptococcus.

The protein of the present invention is a mutant of the wild type protein G · B1 domain, and is compared to a protein comprising the wild type protein G · B1 domain (amino acid sequence of SEQ ID NO: 1 described in Patent Document 8). One or several amino acids in the amino acid sequence of a conventional improved protein having reduced binding in the weakly acidic region of binding to the Fab region of immunoglobulin G and / or binding to the Fc region Those containing mutants obtained by mutation are preferred. As an example of such a conventional improved protein having reduced binding in a weakly acidic region, an improved protein of the wild type protein G • B1 domain described in Patent Document 8 (SEQ ID NOS: 13 to 20). Amino acid sequence). The amino acid sequences of these wild-type protein G · B1 domains and mutants thereof are shown as SEQ ID NOS: 30 to 38 in the sequence listing of the present specification.

Such a mutation in the amino acid sequence of the improved protein can be performed by any method known to those skilled in the art, for example, as described in the examples of the present specification.

As a suitable example of the amino acid sequence of the mutant obtained by further mutating the amino acid sequence of such an improved protein, the amino acids at positions 25, 36 and 41 in the sequence of the protein G / B domain or its domain mutant Mention may be made of proteins in which at least one of the residues is mutated to histidine.

The protein of the present invention can be a tandem multimer of these proteins. The multimer can be appropriately converted into, for example, a dimer, trimer, tetramer, or pentamer according to the wild type. Furthermore, each extracellular domain variant which comprises the multimer contained in the protein of this invention is mutually different, or is mutually the same.

Further, each domain variant may be linked by a linker sequence. Such a linker sequence can be appropriately designed and adjusted by those skilled in the art in consideration of the amino acid sequence of each variant.

The protein of the present invention may be a fusion protein comprising a fused amino acid sequence in which the amino acid sequence of any other protein is linked to the N-terminal side or C-terminal side. Examples of other amino acid sequences used for such fusion proteins include the amino acid sequence of oxaloacetatetdecarboxylase alpha-subunit c-terminal domain (OXADac). In this case, the OXADac-protein G mutant fusion protein can carry a plurality of functions of an avidin binding activity derived from the OXADac region and an antibody binding activity derived from the protein G variant region as a single molecule.

For example, when the protein of the present invention is synthesized in the form of a His-tagged or fusion protein with another protein, the protein is sequenced between the tag and the mutant protein after synthesis or between the other protein and the protein of the present invention. Even if it is degraded with a specific proteolytic enzyme, one to several amino acid residues may remain on the N-terminal side or C-terminal side of the protein of the present invention. In production, methionine derived from an initiation codon may be added to the N-terminal side, but the addition of these amino acid residues does not change the activity of the protein of the present invention as shown below. In addition, the addition of these amino acid residues does not lose the effect of the designed mutation. Therefore, the protein of the present invention naturally includes these mutations. In order to produce the protein of the present invention without addition of such amino acid residues, for example, a protein produced using Escherichia coli or the like, and further using an enzyme such as methionylaminopeptidase, It can be obtained by selectively cleaving amino acid residues (Reference Document 7) and separating and purifying the reaction mixture by chromatography or the like.

In the present specification, including the above proteins, tandem multimers and fusion proteins, they are also simply referred to as “proteins”. The present invention further relates to a nucleic acid encoding the protein, a recombinant vector containing the nucleic acid, and a transformant into which the recombinant vector has been introduced.

In addition, the above-mentioned protein is immobilized on a water-insoluble solid phase support, and is characterized by immunoglobulin G or a protein having an Fc region or Fab region of immunoglobulin G (also referred to as “immunoglobulin G etc.”) ), An antibody containing the capture agent, affinity chromatography for purifying immunoglobulin G and the like, a method for purifying immunoglobulin G and the like using the affinity chromatography for purification, and the like. Here, “having affinity” means that, for example, immunoglobulin G can be adsorbed in chromatography.

In the purification method of the present invention, in order to use the above-mentioned protein as an antibody-supplementing agent, a filler formed by immobilizing a protein on a water-insoluble carrier (water-insoluble solid support) represented by agarose beads is glass. It is preferable to use an affinity column packed in a column such as a tube.

As the adsorption buffer, a buffer having a neutral pH is used, and any salt species can be used as long as the pH can be adjusted. Typically, phosphate buffer, Tris buffer, sodium chloride, etc. The one in which the electrolyte is dissolved is used. The pH of the adsorption buffer is 9.0 to 6.5, preferably pH 8.0 to 7.0. The elution buffer may be in the pH range where the target immunoglobulin G or the like is eluted, and one having a pH of 6.5 to 2.0 is used. The type of elution buffer may be any known to those skilled in the art, and representative examples include phosphate buffer, citrate buffer, acetate buffer, glycine buffer, and the like.

The operation itself in the purification method of the present invention can be carried out by ordinary operations known to those skilled in the art. That is, as in normal affinity purification, first, a sample solution containing immunoglobulin G or the like to be purified is injected into a column stabilized with an adsorption buffer, and immunoglobulin G or the like is adsorbed to the filler. Thereafter, after washing away non-adsorbed components remaining in the column with the adsorption buffer, the immunoglobulin G adsorbed with the elution buffer is eluted, and the immunoglobulin G is recovered in the eluate. It should be noted that other affinity purification conditions such as the flow rate (flow rate) of the adsorption buffer and elution buffer and the column temperature can be appropriately determined by those skilled in the art.

As long as the sample solution contains immunoglobulin G or the like such as serum or ascites culture solution, its origin and other components are not limited. Furthermore, in the purification method of the present invention, any purification means can be used as long as it utilizes the affinity between a protein containing a domain mutant (artificially mutated domain) and immunoglobulin G or the like. In addition to the method to be used, any means known to those skilled in the art such as immunoprecipitation or immobilizing the protein on magnetic beads can be used.

As a preferable example of the mutant of the wild type protein G · B1 domain contained in the improved protein used as the basis for obtaining a suitable protein in the present invention, the mutant protein described in Patent Document 8 is mentioned. be able to. Such mutant proteins can be easily prepared by those skilled in the art according to the method described in Patent Document 7 or Patent Document 8, for example, by the following method.

1. Production of protein (1) Production of protein by genetic engineering a. Gene encoding protein (variant) In the present invention, a genetic engineering method can be used to produce the above-designed protein.
The gene used in such a method has a binding activity to a protein having an Fc region of immunoglobulin G, and at least to an Fc region of immunoglobulin G compared to a protein comprising a wild type protein G / B domain. The binding activity in the weakly acidic region is reduced, while the binding activity in the neutral region to immunoglobulin G is not reduced, such as any of SEQ ID NOS: 14 to 16 in Patent Document 8. Or a variant consisting of an amino acid sequence in which one or several amino acid residues other than the mutated amino acid are deleted, substituted, inserted or added in the amino acid sequence, And a nucleic acid encoding a protein having the characteristics of the present invention.

The gene used in the present invention is a nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising a sequence complementary to the base sequence of the above nucleic acid, and an antibody, immunoglobulin G, or immunoglobulin G. The binding activity to the Fab region of immunoglobulin G and / or the weakly acidic region with respect to the Fc region compared to the corresponding wild-type protein G / extracellular domain protein. Examples also include a nucleic acid encoding the mutant protein having a reduced binding activity. Here, stringent conditions refer to conditions in which a specific hybrid is formed and a non-specific hybrid is not formed. For example, it refers to conditions under which nucleic acids having high identity (identity is 60% or higher, preferably 80% or higher, more preferably 90% or higher, most preferably 95% or higher) hybridize. More specifically, it refers to conditions under which the sodium concentration is 150 to 900 mM, preferably 600 to 900 mM, and the temperature is 60 to 68 ° C., preferably 65 ° C. For example, when hybridization conditions are 65 ° C. and washing conditions are 0.1 × SSC containing 0.1% SDS at 65 ° C. for 10 minutes, hybridization is performed by a conventional method such as Southern blotting or dot blot hybridization. When it is confirmed that the hybridization occurs, it can be said that the cells hybridize under stringent conditions.

The gene encoding the protein of the present invention includes the above nucleic acids and nucleic acids encoding any of the above linker sequences, depending on the desired structure of the protein of the present invention. Nucleic acid encoding each mutant protein constituting a tandem multimer and a nucleic acid encoding a linker sequence may be linked in plural, or the nucleic acid and a nucleic acid encoding an amino acid sequence of an arbitrary protein may be linked. And may be designed to encode a fused amino acid sequence.

b. Gene, Recombinant Vector and Transformant The gene of the present invention described above can be synthesized by chemical synthesis, PCR, cassette mutagenesis, site-directed mutagenesis and the like. For example, a plurality of oligonucleotides up to about 100 bases having a complementary region of about 20 base pairs at the end are chemically synthesized, and the target gene is totally synthesized by combining these and performing the overlap extension method (Reference Document 8). be able to.
The recombinant vector of the present invention can be obtained by linking (inserting) a gene containing the above-described base sequence to an appropriate vector. The vector used in the present invention is not particularly limited as long as it can be replicated in the host or can integrate the target gene into the host genome. For example, bacteriophage, plasmid, cosmid, phagemid and the like can be mentioned.

As plasmid DNA, plasmids derived from actinomycetes (eg pK4, pRK401, pRF31 etc.), plasmids derived from E. coli (eg pBR322, pBR325, pUC118, pUC119, pUC18 etc.), plasmids derived from Bacillus subtilis (eg pUB110, pTP5 etc.) Yeast-derived plasmids (eg, YEp13, YEp24, YCp50, etc.) and the like, and phage DNAs include λ phage (λgt10, λgt11, λZAP, etc.). Furthermore, animal viruses such as retrovirus or vaccinia virus, and insect virus vectors such as baculovirus can also be used.
In order to insert a gene into a vector, first, a method in which purified DNA is cleaved with a suitable restriction enzyme, inserted into a restriction enzyme site or a multicloning site of a suitable vector DNA, and linked to the vector is employed. . The gene needs to be integrated into the vector so that the mutant protein of the invention is expressed. Therefore, the vector of the present invention includes a promoter, a base sequence of a gene, a cis element such as an enhancer, a splicing signal, a poly A addition signal, a selection marker, a ribosome binding sequence (SD sequence), an initiation codon, a termination codon, if desired. Etc. can be connected. A tag sequence for facilitating purification of the protein to be produced can also be linked. As the tag sequence, a base sequence encoding a known tag such as His tag, GST tag, MBP tag, BioEase tag can be used.

Whether or not a gene has been inserted into a vector can be confirmed using a known genetic engineering technique. For example, in the case of a plasmid vector or the like, it can be confirmed by subcloning the vector using a competent cell, extracting the DNA, and then specifying the base sequence using a DNA sequencer. For other vectors that can be subcloned using bacteria or other hosts, the same technique can be used. In addition, vector selection using a selection marker such as a drug resistance gene is also effective.
A transformant can be obtained by introducing the recombinant vector of the present invention into a host cell so that the mutant protein of the present invention can be expressed. The host used for transformation is not particularly limited as long as it can express a protein or polypeptide. Examples include bacteria (E. coli, Bacillus subtilis, etc.), yeast, plant cells, animal cells (COS cells, CHO cells, etc.) and insect cells.

When a bacterium is used as a host, the recombinant vector is autonomously replicable in the bacterium, and at the same time, it is composed of a promoter, a ribosome binding sequence, a start codon, a nucleic acid encoding the mutant protein of the present invention, and a transcription termination sequence. It is preferable. Examples of E. coli include Escherichia coli BL21, and examples of Bacillus subtilis include Bacillus subtilis. The method for introducing a recombinant vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria. Examples thereof include a heat shock method, a method using calcium ions, and an electroporation method.
When yeast is used as a host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe and the like are used. The method for introducing a recombinant vector into yeast is not particularly limited as long as it is a method for introducing DNA into yeast, and examples thereof include an electroporation method, a spheroplast method, and a lithium acetate method.

When animal cells are used as hosts, monkey cells COS-7, Vero, Chinese hamster ovary cells (CHO cells), mouse L cells, rat GH3, human FL cells and the like are used. Examples of methods for introducing a recombinant vector into animal cells include an electroporation method, a calcium phosphate method, and a lipofection method.
When insect cells are used as hosts, Sf9 cells and the like are used. Examples of the method for introducing a recombinant vector into insect cells include the calcium phosphate method, lipofection method, electroporation method and the like.

Whether or not a gene has been introduced into the host can be confirmed by PCR, Southern hybridization, Northern hybridization, or the like. For example, DNA is prepared from the transformant, PCR is performed by designing a DNA-specific primer. Subsequently, the PCR amplification product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, or the like, stained with ethidium bromide, SyberGreen solution, etc., and the amplification product is detected as a single band. You can confirm that it has been converted. Moreover, PCR can be performed using a primer previously labeled with a fluorescent dye or the like to detect an amplification product.

c. Obtaining Protein by Transformant Culture When producing the protein as a recombinant protein, the protein of the present invention can be obtained by culturing the above-described transformant and collecting it from the culture. The culture means any of culture supernatant, cultured cells, cultured cells, or disrupted cells or cells. The method for culturing the transformant of the present invention is carried out according to a usual method used for culturing a host.

A medium for culturing transformants obtained using microorganisms such as Escherichia coli and yeast as a host contains a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by the microorganisms, and efficiently cultures the transformants. As long as the medium can be used, either a natural medium or a synthetic medium may be used. Examples of the carbon source include carbohydrates such as glucose, fructose, sucrose, and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol. Examples of the nitrogen source include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium salts of organic acids such as ammonium phosphate or other nitrogen-containing compounds, peptone, meat extract, corn steep liquor, and the like. Examples of the inorganic substance include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate. The culture is usually performed at 20 to 37 ° C. for 12 hours to 3 days under aerobic conditions such as shaking culture or aeration and agitation culture.

When the protein of the present invention is produced in cells or cells after culturing, the protein or cells are collected by crushing the cells or cells by sonication, repeated freeze-thawing, homogenizer treatment, etc. . When the protein is produced outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like. Thereafter, a general biochemical method used for protein isolation and purification, for example, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc. can be used alone or in appropriate combination in the culture. From the above, the protein of the present invention can be isolated and purified.

In addition, when using a so-called cell-free synthesis system in which only factors (enzymes, nucleic acids, ATP, amino acids, etc.) involved in protein biosynthesis reactions are mixed, the mutant protein of the present invention can be obtained from a vector without using living cells. Can be synthesized in vitro (Reference 9). Thereafter, using the same purification method as described above, the mutant protein of the present invention can be isolated and purified from the mixed solution after the reaction.
In order to confirm whether the isolated and purified protein of the present invention is a protein having a desired amino acid sequence, a sample containing the protein is analyzed. As an analysis method, SDS-PAGE, Western blotting, mass spectrometry, amino acid analysis, amino acid sequencer, etc. can be used (Reference Document 10).

(2) Production of protein by other methods The protein of the present invention can also be produced by organic chemical methods such as solid phase peptide synthesis. Protein production methods using such techniques are well known in the art and are briefly described below.
When a protein is chemically produced by a solid phase peptide synthesis method, the protection having the amino acid sequence of the protein of the present invention is preferably achieved by repeating the polycondensation reaction of the activated amino acid derivative using an automatic synthesizer. The polypeptide is synthesized on the resin. Next, the protective polypeptide is cleaved from the resin and the side chain protecting group is cleaved simultaneously. This cleaving reaction is known to have an appropriate cocktail depending on the type of resin, protecting group, and amino acid composition (Reference 11). Thereafter, the crude protein is transferred from the organic solvent layer to the aqueous layer, and the target protein is purified. As a purification method, reverse phase chromatography or the like can be used (Ref. 11).

2. Protein Immobilization The protein of the present invention can be used as a capture agent for antibodies and the like by utilizing its antibody binding property. The antibody capture agent can be used for purification and removal of antibodies, diagnosis, treatment, examination, etc. using antibodies.
The antibody capture agent of the present invention may be in any form as long as it contains the protein of the present invention. Preferably, the form of the mutant protein of the present invention immobilized on a water-insoluble solid support. Is appropriate. Examples of the water-insoluble carrier used include inorganic carriers such as glass beads and silica gel, synthetic polymers such as crosslinked polyvinyl alcohol, crosslinked polyacrylate, crosslinked polyacrylamide, and crosslinked polystyrene, crystalline cellulose, crosslinked cellulose, crosslinked agarose, and crosslinked dextran. Examples include organic carriers composed of polysaccharides, and organic-organic and organic-inorganic composite carriers obtained by a combination thereof. Among them, hydrophilic carriers have relatively little nonspecific adsorption, and antibodies or immunoglobulin G Alternatively, it is preferable because the protein having the Fc region of immunoglobulin G has good selectivity. The term “hydrophilic carrier” as used herein refers to a carrier having a contact angle with water of 60 ° or less when the compound constituting the carrier is formed into a flat plate shape. Such carriers include cellulose, chitosan, dextran and other polysaccharides, polyvinyl alcohol, saponified ethylene-vinyl acetate copolymer, polyacrylamide, polyacrylic acid, polymethacrylic acid, polymethyl methacrylate, polyacrylic acid grafting Representative examples include carriers made of polyethylene, polyacrylamide grafted polyethylene, glass and the like.

Commercially available products include porous cellulose gels GCL2000 and GC700, Sephacryl® S-1000 with allyl dextran and methylenebisacrylamide cross-linked covalently, acrylate-based carrier Toyopearl, agarose-based cross-linked carrier SepharoseCL4B, epoxy group For example, Eupergit C250L, which is polymethacrylamide activated by the above method, can be exemplified. However, the present invention is not limited to these carriers and activated carriers. Each of the above carriers may be used alone, or any two or more of them may be mixed. The water-insoluble carrier used in the present invention preferably has a large surface area in view of the purpose and method of use of the present antibody capture agent, and has a large number of pores of an appropriate size, that is, is porous. preferable.

The form of the carrier can be any of beads, fibers, membranes (including hollow fibers), and any form can be selected. A bead shape is particularly preferably used because of easy preparation of a carrier having a specific exclusion limit molecular weight. The average particle size of beads is 10 to 2500 μm, and is particularly preferably in the range of 25 μm to 800 μm from the viewpoint of easy ligand immobilization reaction.
Furthermore, if a functional group that can be used for the ligand immobilization reaction is present on the surface of the carrier, it is convenient for immobilization of the ligand. Representative examples of these functional groups include hydroxyl group, amino group, aldehyde group, carboxyl group, thiol group, silanol group, amide group, epoxy group, succinimide group, acid anhydride group, iodoacetyl group and the like.
In immobilization of the mutant protein to the carrier, it is immobilized via a hydrophilic spacer in order to improve the capture efficiency by reducing the steric hindrance of the mutant protein and to suppress non-specific binding. It is more preferable. As the hydrophilic spacer, for example, a polyalkylene oxide derivative in which both ends are substituted with a carboxyl group, an amino group, an aldehyde group, an epoxy group or the like is preferably used.

The method and conditions for immobilizing the mutant protein to be introduced into the carrier and the organic compound used as the spacer are not particularly limited, but the method generally employed when immobilizing a protein or peptide on the carrier is used. Illustrate. The carrier is reacted with cyanogen bromide, epichlorohydrin, diglycidyl ether, tosyl chloride, tresyl chloride, hydrazine, etc. to activate the carrier (compounds that are immobilized as ligands from the functional groups that the carrier originally has reacted) A functional group that can be easily immobilized), reacting with a compound to be immobilized as a ligand, a method of immobilizing, or a system in which a compound to be immobilized as a carrier and a ligand exists, such as a condensation reagent such as carbodiimide, or glutaraldehyde In addition, there is an immobilization method by adding a reagent having a plurality of functional groups in the molecule to condense and crosslink, but the immobilization method does not easily desorb proteins from the carrier when the capture agent is sterilized or used. It is more preferable to apply.

3. Performance confirmation test of protein and antibody capture agent The protein produced as described above (hereinafter also simply referred to as “protein”) and the antibody capture agent may be selected by performing the following performance confirmation test and selecting a good one. However, both the protein and the antibody capturing material of the present invention had good performance.

(1) Antibody binding test The antibody binding of the protein of the present invention is confirmed and evaluated using Western blotting, immunoprecipitation, pull-down assay, ELISA (Enzyme-Linked ImmunoSorbent Assay), surface plasmon resonance (SPR) method, etc. can do. In particular, the SPR method allows the interaction between living organisms to be observed over time in real time without a label, so that the binding reaction of the mutant protein can be quantitatively evaluated from a kinetic viewpoint.
Moreover, the antibody binding property of the mutant protein immobilized on the water-insoluble solid phase support can be confirmed and evaluated by the above SPR method or liquid chromatography method. Among them, the liquid chromatography method can accurately evaluate the pH dependence on the antibody binding property.
(2) Thermal stability test of protein The thermal stability of the mutant protein of the present invention includes circular dichroism (CD) spectrum, fluorescence spectrum, infrared spectroscopy, differential scanning calorimetry, residual activity after heating. Etc. can be used for evaluation. In particular, the CD spectrum is a spectroscopic analysis method that sharply reflects changes in the secondary structure of the protein, so the change in the three-dimensional structure with respect to the temperature of the mutant protein is observed, and the structural stability is quantified thermodynamically. Can be evaluated.

[References]
Reference 1; Bjorck L, Kronvall G. (1984) Purification and some properties of streptococcal protein G, a novel IgG-binding reagent. J Immunol. 133, 69-74.
Reference 2; Boyle M. DP, Ed. (1990) Bacterial Immunoglobulin Binding Proteins. Academic Press, Inc., San Diego, CA.
Reference 3; Gallagher T, Alexander P, Bryan P, Gilliland GL. (1994) Two crystal structures of the B1 immunoglobulin-binding domain of streptococcal protein G and comparison with NMR. Biochemistry 19, 4721-4729.
Reference 4; Sauer-Eriksson AE, Kleywegt GJ, Uhlen M, Jones TA. (1995) Crystal structure of the C2 fragment of streptococcal protein G in complex with the Fc domain of human IgG. Structure 3, 265-278.
Reference 5; Derrick JP, Wigley DB. (1994) The third IgG-binding domain from streptococcal protein G. An analysis by X-ray crystallography of the structure alone and in a complex with Fab. J Mol Biol. 243, 906- 918.
Reference 6; Alexander P, Fahnestock S, Lee T, Orban J, Bryan P. (1992) Thermodynamic analysis of the folding of the streptococcal protein G IgG-binding domains B1 and B2: why small proteins tend to have high denaturation temperatures. Biochemistry 14, 3597-3603.
Reference 7; D'souza VM, Holz RC. (1999) The methionyl aminopeptidase from Escherichia coli can function as an iron (II) enzyme. Biochemistry 38, 11079-11085.
Reference 8; Horton R. M., Hunt H. D., Ho S. N., Pullen J. M. and Pease L. R. (1989). Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77, 61-68.
Reference 9; Masato Okada, Kaoru Miyazaki (2004) Protein Experiment Note (above), Yodosha Reference 10; Shigeo Ohno, supervised by Yoshifumi Nishimura (1997) Protein Experiment Protocol 1-Functional Analysis, Shujunsha Reference 11 Supervised by Shigeo Ohno and Yoshifumi Nishimura (1997) Protein Experiment Protocol 2-Structural Analysis, Shujunsha

The following contents are specifically disclosed in Patent Document 8.

In Example 1, the wild type amino acid sequence of the protein G • B1 domain represented by [SEQ ID NO: 1], the wild type amino acid sequence of the protein G • B2 domain represented by [SEQ ID NO: 2], and [SEQ ID NO: 3] Based on the wild-type amino acid sequence of the protein G / B3 domain shown, a mutant protein contained in the protein used in the present invention in which a mutation is introduced into the B1, B2 or B3 domain of protein G (hereinafter referred to as "improved protein G") The amino acid residue to be substituted is specified and the amino acid residue to be substituted is specified.

In Example 2, the amino acid sequences of a plurality of improved protein Gs represented by [SEQ ID NO: 4] to [SEQ ID NO: 19] are obtained using the selected mutation target site and the above-identified amino acid residue information to be substituted. In addition, [SEQ ID NO: 13] to [SEQ ID NO: 20] were finally selected as specific amino acid sequences, and an improved protein G showing this sequence was actually synthesized and its molecular properties were evaluated. Are listed.

In Example 3, the base sequence of the nucleic acid encoding the amino acid sequence of the improved protein G ([SEQ ID NO: 13] to [SEQ ID NO: 20]) and the base sequence of Oxaloacetate decarboxylase alpha-subunit c-terminal domain (OXADac) No. 31] The nucleotide sequence of the mutant protein using cocoons is described.

In Example 4, a plasmid vector containing a gene encoding improved protein G was synthesized using the PG gene consisting of the nucleotide sequence of [SEQ ID NO: 21] to [SEQ ID NO: 29], and then Oxaloacetate decarboxylase using E. coli. It describes the production of a fusion protein of alpha-subunit c-terminal domain (OXADac) [SEQ ID NO: 31] and a mutant protein.

In Example 5, a plasmid vector containing a gene encoding improved protein G was synthesized using various primers ([SEQ ID NO: 32] to [SEQ ID NO: 35]), and then Met-added improved protein using Escherichia coli. The manufacture of G is described.

In Example 6, the purity of the improved protein G was confirmed by polyacrylamide gel electrophoresis. In Example 7, the molecular weight of the improved protein G was measured by a MALDI-TOF mass spectrometer. In Example 8, the protein was identified. In Example 8, pH gradient affinity chromatography was performed using a column on which the OXADac-PG fusion protein was immobilized, and the pH at which the monoclonal antibody was eluted was examined. In Example 9, in Example 9, stepwise pH affinity chromatography was performed using a column immobilized with OXADac-PG fusion protein, and elution of monoclonal antibodies was examined at several pHs. In Example 10, the protein dissociation property in the weakly acidic region of improved protein G was evaluated. In G mutant) was evaluated by surface plasmon resonance (SPR) method. In Example 11, the mutant was obtained in the neutral region and in the weakly acidic region where 95% or more of the histidine residues were protonated. The antibody binding property of the protein was evaluated by the surface plasmon resonance (SPR) method, the thermal stability of the mutant protein was evaluated in Example 12, the single crystal of the mutant protein was prepared in Example 13, It is described that the three-dimensional structure was determined by X-ray diffraction analysis.

Furthermore, in Example 14, a tandem type of a trimeric wild type PG (CGB01H-3D, [SEQ ID NO: 36]) し た added with a cysteine residue and a His tag on the carboxyl terminal side or a mutant PG which is a protein of the present invention. Production of a tandem multimer of an extracellular domain mutant of protein G using two artificially synthesized plasmids incorporating a spider gene encoding a trimer (CGB19H-3D, [SEQ ID NO: 37]) and the protein It describes the preparation of the affinity chromatography column to be used and the purification of human IgG1 antibody and IgG3 antibody using the column.

Example 15 describes a comparison of protein G extracellular domain mutant tandem multimers and the same monomers with respect to antibody binding dissociation with IgG1 type humanized monoclonal antibodies.

In Example 16, a mutant PG monomer mutant PG (CGB19H-1D, FIG. 4, [SEQ ID NO: 38]) having a cysteine residue and a His tag added to the carboxyl terminus, a tandem tetramer of the mutant PG Incorporating a gene encoding the body PG (CGB19H-4D, FIG. 4, [SEQ ID NO: 39]) and the tandem pentamer PG of the mutant PG (CGB19H-5D, FIG. 4, [SEQ ID NO: 40]) 3 It describes the production of a protein G extracellular domain mutant monomer, tandem tetramer, and pentamer using a species of artificially synthesized expression plasmid.

In Example 17, each of the mutant proteins for the IgG1-type humanized monoclonal antibody was prepared by immobilizing the monomer and tandem-type multimer of the extracellular domain mutant of protein G to the solid phase through a cysteine residue at the carboxyl terminal. It is described that the antibody binding property of each was comparatively evaluated by the SPR method.

Hereinafter, the present invention will be specifically described with reference to examples. However, the technical scope of the present invention is not limited to these examples. In the present specification, various amino acid residues are described by the following abbreviations. Ala; L-alanine residue, Arg; L-arginine residue, Asp; L-aspartic acid residue, Asn; L-asparagine residue, Cys; L-cysteine residue, Gln; L-glutamine residue, Glu L-glutamic acid residue, Gly; L-glycine residue, His; L-histidine residue, Ile; L-isoleucine residue, Leu; L-leucine residue, Lys; L-lysine residue, Met; L -Methionine residue, Phe; L-phenylalanine residue, Pro; L-proline residue, Ser; L-serine residue, Thr; L-threonine residue, Trp; L-tryptophan residue, Tyr; L-tyrosine Residue, Val; L-valine residue. In the present specification, the amino acid sequence of a peptide is described according to a conventional method such that the amino terminus (hereinafter referred to as N-terminus) is located on the left side and the carboxyl terminus (hereinafter referred to as C-terminus) is located on the right side.

In this example, the protein (variant) of the present invention was produced. These were reported by Watanabe et al. (Non-patent Document 5), such as histidine residues of a fusion protein (SEQ ID NO: 1) containing an extracellular domain mutant of protein G (hereinafter referred to as “modified protein G”). It consists of an amino acid sequence in which a part is substituted with a wild-type amino acid residue.

With the expectation that electrostatic repulsion between residues occurs in the protein G molecule in an acidic solution, the site of mutagenesis introduction of histidine was determined as follows. The lysine residues that are positively charged residues of the improved protein G are identified from the three-dimensional structure information (PDB: 2ZW1) of protein G downloaded from the public database Protein Data Bank, and the residues present within 7 か ら of the lysine residue Was identified as a candidate for mutagenesis. For the calculation, CONTACT of the structural analysis software CCP4 (Non Patent Literature 6) was used. Of these residues within 7%, residues that have been found to be important for binding to Fc from the previously reported mutant analysis (Non-Patent Document 7), and the structure of protein G that has been determined from structural information Residues important for formation were excluded from the mutagenesis candidates, and residues that contact these important residues were also excluded from the candidates. As a result, nine residue positions of Asp1, Asn8, Glu15, Thr25, Glu36, Tyr33, Gly41, Thr55, and Glu56 were determined as mutation introduction sites. In relation to the binding interface with the Fc region, these are in the vicinity of the binding interface (that is, the corresponding lysine residue is within 6 angstroms from any amino acid residue in the Fc region) (Thr25, Glu36, Gly41, Thr55) and other groups far from the bonding interface (Asp1, Asn8, Glu15, Tyr33, 遠 Glu56). Various information regarding the residues into which the above mutations have been introduced is shown in Table 1 below.

Sense and antisense oligo DNA primers containing a histidine codon for an expression plasmid containing a fusion protein gene (SEQ ID NO: 2) containing the improved protein G (PG19) produced by the method of Watanabe et al. (SEQ ID NOs: 3 to 20), site-directed mutagenesis PCR reaction and template plasmid cleaving reaction (FIG. 1) were performed, and a histidine residue was substituted at the mutagenesis site determined by the above method. Mutant gene encoding the amino acid sequence (SEQ ID NO: 21-29) of the fusion protein containing the body (PG19 T25H, PG19 Y33H, PG19 E36H, PG19 G41H, PG19 T55H, PG19 D1H, PG19 N8H, PG19 E15H, Each was produced. An expression plasmid containing the gene of each histidine additional mutant was isolated and purified, and transformed into E. coli BL21 (DE3) strain (Novagen) for expression. The pre-cultured transformant was subcultured to 2YT medium at 2 ml / 200 ml, and cultured with shaking until O.D.600 = 0.8-1.0. In order to express the target protein, 0.5 mM IPTG was added, and further cultured with shaking at 37 ° C. for 2 hours. The collected microbial cells were suspended in 10 mL of PBS, subjected to ultrasonic disruption and then sterilized by filtration to obtain a total protein solution. The histidine additional mutant was adsorbed onto a Ni Sepharose (GE ealthcare) 2 ml column, washed with 20 mM imidazole, and eluted with 500 mM imidazole to obtain a purified protein. Improved protein G (PG19) was prepared in the same manner using the corresponding expression plasmid.

In this example, the histidine additional mutant of the present invention was immobilized on a solid phase via a cysteine residue at the carboxyl terminus, and the pH responsiveness of each histidine additional mutant was evaluated by the SPR method. For comparison, improved protein G was also evaluated in the same manner.

First of all, the improved protein G (PG19) and histidine additional mutant (PG19 T25H, PG19 Y33H, PG19 E36H, PG19 G41H, PG19 T55H, PG19 D1H, PG19 N8H, PG19 E15H and PG19 E56H) were immobilized by maleimide coupling method using EMCH (N- [ε-Maleimidocaproic acid] hydrazide, trifluoroacetic acid) (Thermo scientific). Subsequently, IgG1-type humanized monoclonal antibody was dissolved in a running buffer (10 mM HEPES pH7.4, 150 mM NaCl, 0.005% v / v Surfactant P20) to prepare a sample antibody solution of 1 mg / ml. SPR measurement was performed using a Biacore T100 (GE Healthcare) at a reaction temperature of 25 ° C., and the SPR response at each pH solution was measured. (Figures 2-4).

From the sensorgram, the antibody dissociation rate and leak rate in pH 4 solution were calculated and the pH responsiveness was compared. The results are shown in Table 2 below. Since PG19 G41H, which showed the highest dissociation rate at pH 4, had already leaked most of the antibody before adding the pH 4 solution, it was found that this mutant had reduced antibody binding ability. Next, it was found that PG19 T25H, which showed the next highest dissociation rate, had no decrease in binding ability, and the response at pH で 4 was significantly increased compared to PG19.

In Table 2, the SPR response immediately after the addition of the antibody is dissociation rate = 0%, and the SPR response after completely removing the antibody with the regeneration solution is the dissociation rate = 100%. %) ”. “Leak (%)” was calculated from the decrease in response in the buffer before the addition of the pH 4 solution. In addition, please also refer FIG. 5 regarding a reference | standard etc. in calculation of the dissociation rate (%) and leak (%) from a sensorgram.

In this example, an extracellular domain mutant of protein G was produced, and a column using the protein was produced.
(1) Expression and purification of recombinant PG Fusion protein comprising mutants (PG19, PG19 T25H, PG19 E36H, PG19 G41H) constructed in Example 1 and wild-type protein G (PG01) described in Non-Patent Document 5 Escherichia coli transformed with an expression plasmid containing the gene of SEQ ID NO: 38) was subcultured in LB medium and cultured with shaking until OD600 = 0.8 to 1.0. To express the target protein, 0.5 mM IPTG was added, and further cultured with shaking at 37 ° C. for 3 hours. The collected cells were suspended in PBS, subjected to ultrasonic disruption and then centrifuged, and the resulting supernatant was used as a total protein solution. Recombinant PG was adsorbed onto a 1 ml column of HisTrap FF (GE Healthcare Bioscience), washed with 20 mM imidazole, and eluted with 500 mM imidazole to obtain purified protein.

(2) Immobilization of recombinant PG and column preparation
Sepharose 4 Fast Flow (GE Healthcare) was filtered off with a glass filter and washed with ultrapure water to obtain 10 ml of a carrier. The carrier was transferred to the flask, 3 ml of 2M aqueous sodium hydroxide solution and 4 g of butanediol diglycidyl ether were added, and the mixture was reacted by shaking at 25 ° C. for 4 hours. It was filtered with a glass filter and washed with ultrapure water to obtain an activated carrier. 1 mL of the activated carrier was collected on a glass filter and washed with a coupling buffer (0.1 M sodium phosphate, 1.0 M sodium sulfate, 1 mM EDTA, pH 8.0). Transfer the activated carrier to the flask, add 1.4 ml of recombinant PG-containing solution containing 5 mg / ml recombinant PG (PG19 T25H) and 2 mL of coupling buffer, and shake for 16 hours at 37 ° C and 150 rpm. Of cysteine residues. The solution was filtered with a glass filter and washed with a coupling buffer. Next, the carrier was transferred to a flask, 3 ml of a solution of 1 M thioglycerol, 0.1 M sodium phosphate, 1 mM EDTA, pH 8.0 was added, and the mixture was shaken at 37 ° C. and 150 rpm for 4 hours to mask unreacted active groups. The solution was filtered off with a glass filter, and washing solution 1 (0.1 M Tris-HCl, 0.5 M sodium chloride, pH 8.0) and washing solution 2 (0.1 M acetic acid, 0.5 M sodium chloride, pH 4.0) were alternately washed with 15 ml for 3 cycles. 1 ml of the immobilization carrier was washed with ultrapure water and packed in a Tricon 5/50 Column.

For other recombinant PGs, columns were prepared in the same manner as PG19 T25H. The amount of PG containing liquid is as follows.
・ PG19 E36H: 921μL of 7.6mg / mL recombinant PG-containing solution
・ PG19 G41H: 897μL of 7.8mg / mL recombinant PG solution
-PG01: 853μL of 8.2mg / mL recombinant PG containing solution
・ PG19: 1.4mL of 12.0mg / mL recombinant PG solution

In this example, the antibody adsorption capacity of the recombinant PG-immobilized column prepared in Example 3 was measured.
Set the recombinant PG-immobilized column in the liquid chromatography device AKTAexplore (GE Healthcare Bioscience) and add the adsorption buffer (20 mM phosphate buffer, 150 mM sodium chloride, pH 7.2) at 1 mL / min or 0.4 mL / min. After flowing and equilibrating under conditions, human IgG (oriental yeast) prepared to 1 mg / mL was injected. The injection was continued until the absorbance at 280 nm of the eluate reached 15% of the absorbance of the injected sample, and after washing with the adsorption buffer, the adsorption buffer was replaced with 20 mM citric acid (pH 2.4).

The dynamic binding capacity (DBC) was calculated from the amount of sample injected until the absorbance excluding non-adsorbed components at 280 nm of the eluate reached 10% of the absorbance of the injected sample. Table 3 shows the DBC of each immobilized column. PG19 G41H had a significantly reduced DBC compared to other columns.

In this example, pH gradient affinity chromatography was performed using the recombinant PG-immobilized column prepared in Example 3.
Set the recombinant PG-immobilized column in the liquid chromatography device AKTAexplore (GE Healthcare Bioscience), and equilibrate by flowing an adsorption buffer solution (20 mM phosphate buffer, 150 mM sodium chloride, pH 7.2) at 1 ml / min. Then, human IgG (oriental yeast) prepared to 1 mg / mL was injected. After washing with an adsorption buffer, the solution was replaced with 20 mM citric acid buffer (pH 6.0) and continuously with 20 mM citric acid (pH 2.4) at a flow rate of 1.0 mL / min over 80 min.

Human IgG elution peak is around pH3.9 for PG01 immobilized column, around pH4.4 for PG19 immobilized column, around pH5.0 for PG19 T25H immobilized column, around pH4.5 for PG19 E36H immobilized column, PG19 G41H It was around pH 6.5 in the immobilized column (FIG. 6). Therefore, the PG19 G41H immobilized column can elute human IgG on the neutral side from pH 6.0, and the PG19 T25H and PG19 E36H immobilized columns elute under mild acidic conditions compared to the PG19 immobilized column. It became clear that it was possible.

Currently, the wild-type protein G extracellular domain is commercially available as an affinity chromatography carrier for antibody purification and a test reagent for antibody detection, and is widely used in each field of life science. In addition, with the recent development of antibody-related industries including antibody drugs, the demand for these products has increased dramatically. Therefore, many protein G extracellular domain-containing products utilize the advantage that the binding activity in the weakly acidic region to the Fc region of immunoglobulin G is reduced by substituting the protein of the present invention with the wild type. This greatly contributes to technological development in a wide range of technical fields dealing with various antibodies.

Claims (19)

1. Compared to the protein consisting of wild-type protein G and B domains, it has binding activity to the Fc region of immunoglobulin G. By substituting residues near the positively charged residues of protein G with positively charged residues A protein with reduced binding activity in the weakly acidic region to the Fc region of immunoglobulin G.
2. The protein according to claim 1, wherein the mutated amino acid residue is histidine.
3. Neighboring residues are within 6 angstroms from any amino acid residue in the Fc region in the wild type protein G / Fc complex crystal structure or the domain variant / Fc complex model structure of wild type protein G derived therefrom The protein according to claim 1 or 2, which is an amino acid residue within 6 angstroms from a positively charged residue of wild-type protein G or a domain variant thereof.
4. The protein according to any one of claims 1 to 3, wherein the wild-type protein G · B domain is any one of B1, B2 and B3 of protein G of Streptococcus spp.
5. The protein according to any one of claims 1 to 4, wherein the wild-type protein G / B domain has an amino acid sequence represented by SEQ ID NO: 30, and a domain variant thereof is represented by any one of SEQ ID NOs: 31 to 38.
6. 6. The protein G / B domain or a domain variant thereof, wherein at least one of amino acid residues at positions 25, 36 and 41 is mutated to histidine. Protein.
7. The protein according to any one of claims 1 to 6, wherein the antibody dissociation rate in the pH4 solution is higher than that of wild-type protein G · B or a domain variant thereof in the measurement by SPR method.
8. A protein that is a tandem multimer of the protein according to any one of claims 1 to 7.
9. The protein according to claim 6, which is a trimer, a tetramer, or a pentamer.
10. The protein according to claim 8 or 9, wherein the extracellular domain mutants constituting the multimer are identical to each other.
11. The protein according to any one of claims 8 to 10, wherein each extracellular domain variant is linked by a linker sequence.
12. A protein which is a fusion protein comprising an amino acid sequence obtained by linking the amino acid sequence of the protein according to any one of claims 1 to 11 and the amino acid sequence of another protein.
13. The protein according to claim 12, which has an amino acid sequence represented by any one of SEQ ID NOs: 21, 23 and 24.
14. A nucleic acid encoding the protein according to any one of claims 1 to 13.
15. A recombinant vector containing the nucleic acid according to claim 14.
16. A transformant into which the recombinant vector according to claim 15 has been introduced.
17. 14. A protein having immunoglobulin F or an immunoglobulin G Fc region or Fab region, wherein the protein according to any one of claims 1 to 13 is immobilized on a water-insoluble solid phase support. Scavenger.
18. Affinity chromatography for purification of immunoglobulin G or a protein having an Fc region or Fab region of immunoglobulin G, comprising the capture agent according to claim 17.
19. A method for purifying immunoglobulin G or a protein having an Fc region or Fab region of immunoglobulin G using the purification affinity chromatography according to claim 18.

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