Patent Description:
Antibody drugs are pharmaceuticals utilizing an antibody (immunoglobulin), which is a molecule responsible for immune functions in living organisms. By virtue of diversity of the variable region of each antibody, antibody drugs are capable of binding to target molecules with high specificity and affinity. Antibody drugs therefore have fewer side effects. Because of this, and the fact that such drugs have become applicable to a wider range of diseases in recent years, the market for antibody drugs has been rapidly expanding.

Production of an antibody drug includes a culture step and a purification step, wherein productivity in the culture step is improved by modification of antibody-producing cells and optimization of culture conditions. The purification step employs affinity chromatography for crude purification. This is followed by intermediate purification, final purification, and then virus removal before the formulation.

In the purification step, an affinity support that specifically recognizes an antibody molecule is used. As the ligand protein to be used for the support, protein A or protein G having the property of binding to the antibody (immunoglobulin) is used. In the production of the antibody drug, the affinity support is used a plurality of times for reduction of the production cost. After using the affinity support, a step of removing impurities remaining on the support is carried out. Usually, in the step of removing impurities remaining on the support, cleaning-in-place using sodium hydroxide is carried out to regenerate the affinity support. Therefore, the ligand protein needs to have sufficient chemical stability so that the antibody-binding capacity can be maintained even after this step.

Examples of the chemically stable ligand protein used for the affinity support include an alkali-stable chromatography ligand using an amino acid sequence of domain C of protein A (SpA) derived from a bacterium belonging to the genus Staphylococcus (Patent Document <NUM>), and an affinity chromatography ligand composed of the same amino acid sequence as domain B, domain C, or domain Z of the protein A except for the presence of partial deletion (Patent Document <NUM>). Further, it is known that substitution of the glycine at position <NUM> of domain Z to alanine stabilizes the structure (Non-patent Document <NUM>).

Document <CIT> discloses SpA C domain mutant G29A/S33E which retains immunoglobulin-binding capacity at <NUM>% after alkali treatment.

An object of the present invention is to provide an immunoglobulin-binding protein having improved stability against alkali.

As a result of intensive study, the present inventors identified amino acid residues involved in improvement of the stability in domain C of protein A (SpA) derived from a bacterium belonging to the genus Staphylococcus, and discovered that, by substituting the amino acid residues to other specific amino acid residues, excellent stability against alkali can be achieved, thereby completing the present invention.

Specifically, the present invention is defined by the claims.

More specifically, the present invention includes the following modes.

An immunoglobulin-binding protein comprising an amino acid sequence which is the same as an amino acid sequence of an immunoglobulin-binding domain of protein A except that the amino acid sequence of the immunoglobulin-binding protein comprises the amino acid sequence of any of SEQ ID NOs:<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> to <NUM>, <NUM>, <NUM>, and <NUM> to <NUM>.

A polynucleotide encoding the immunoglobulin-binding protein.

An expression vector comprising the polynucleotide.

A transformant comprising the polynucleotide or the expression vector.

The transformant which is Escherichia coli.

A method of producing an immunoglobulin-binding protein, the method comprising the steps of:.

A method of separating immunoglobulin, the method comprising the steps of:.

The present invention is described below in detail.

The immunoglobulin-binding protein of the present invention is a particular immunoglobulin-binding protein. The "immunoglobulin-binding protein" means a protein having binding capacity to immunoglobulin. Thus, the immunoglobulin-binding protein of the present invention has binding capacity to immunoglobulin. More specifically, the immunoglobulin-binding protein of the present invention may have binding capacity to the Fc region of immunoglobulin. The binding capacity to immunoglobulin is also referred to as "immunoglobulin-binding activity" or "antibody-binding activity". The immunoglobulin-binding activity can be measured by, for example, the ELISA method. The ELISA method can be carried out under, for example, the conditions described in Examples.

Examples of the immunoglobulin-binding protein of the present invention include a protein containing an amino acid sequence which is the same as an amino acid sequence of an immunoglobulin-binding domain of protein A except that the amino acid sequence of the immunoglobulin-binding protein has an amino acid substitution(s) at a particular position(s). An amino acid sequence of an immunoglobulin-binding domain of protein A having no amino acid substitution(s) at the particular position(s) is also referred to as "unmodified amino acid sequence". An amino acid sequence of an immunoglobulin-binding domain of protein A having the amino acid substitution(s) at the particular position(s) is also referred to as "modified amino acid sequence". In other words, the modified amino acid sequence may be an amino acid sequence which is the same as the unmodified amino acid sequence except that the modified amino acid sequence has the amino acid substitution(s) at the particular position(s). The immunoglobulin-binding protein of the present invention may be, for example, a protein containing an amino acid sequence which is the same as the unmodified amino acid sequence except that the amino acid sequence of the protein has the amino acid substitution(s) at the particular position(s). Further, the immunoglobulin-binding protein of the present invention may be, for example, a protein containing the modified amino acid sequence. The unmodified amino acid sequence may or may not be a naturally occurring amino acid sequence. The unmodified amino acid sequence may be modified, for example, so as to have a desired property. The unmodified amino acid sequence may have, for example, an amino acid substitution(s) other than the amino acid substitution(s) at the particular position(s).

Examples of the protein A include protein A (SpA) derived from a bacterium belonging to the genus Staphylococcus. Examples of the bacterium belonging to the genus Staphylococcus include Staphylococcus aureus. The immunoglobulin-binding domain includes domain C. Examples of the domain C of SpA derived from Staphylococcus aureus include the amino acid residues at positions <NUM> to <NUM> of GenBank No. AAA26676. The amino acid sequence of the domain C is shown in SEQ ID NO: <NUM>. The modified amino acid sequences include an amino acid sequence which is the same as an amino acid sequence of an immunoglobulin-binding domain exemplified above such as the amino acid sequence of SEQ ID NO: <NUM> except that the modified amino acid sequence has the amino acid substitution(s) at the particular position(s). Thus, the immunoglobulin-binding proteins of the present invention include a protein containing an amino acid sequence which is the same as an amino acid sequence of an immunoglobulin-binding domain exemplified above such as the amino acid sequence of SEQ ID NO: <NUM> except that the amino acid sequence of the immunoglobulin-binding protein has the amino acid substitution(s) at the particular position(s). In other words, the immunoglobulin-binding proteins of the present invention are proteins containing the same amino acid sequence as an amino acid sequence of an immunoglobulin-binding domain exemplified above such as the amino acid sequence of SEQ ID NO: <NUM> except for the presence of the amino acid substitution(s) at the particular position(s).

The fact that a protein contains an amino acid sequence is also referred to as "a protein contains amino acid residues having an amino acid sequence". The fact that a protein or an amino acid sequence has an amino acid substitution(s) is also referred to as "an amino acid substitution(s) occur(s) in a protein or an amino acid sequence". The amino acids constituting a protein or an amino acid sequence are also referred to as "amino acid residues".

Disclosed herein but not part of the claimed subject-matter, the amino acid substitution(s) at the particular position(s) is/are at least one amino acid substitution selected from Asp2Glu (this expression represents the fact that the amino acid residue corresponding to the aspartic acid at position <NUM> of SEQ ID NO:<NUM> is substituted to glutamic acid; the same applies hereinafter), Lys49Met, Asn21Tyr, Lys58Glu, Lys58Val, Lys58Gly, Lys58Asp, Asn3Ile, Asn3Thr, Asn11Lys, Asn11Tyr, Glu15Ala, and Val40Ala. Disclosed herein but not part of the claimed subject-matter,, the amino acid substitution(s) at the particular position(s) is/are at least one amino acid substitution selected from (<NUM>) Asp2Glu; (<NUM>) Lys49Met; (<NUM>) Asn21Tyr; (<NUM>) Lys58Glu, Lys58Val, Lys58Gly, or Lys58Asp; (<NUM>) Asn3Ile or Asn3Thr; (<NUM>) Asn11Lys or Asn11Tyr; (<NUM>) Glu15Ala; and (<NUM>) Val40Ala. Among these, Asn21Tyr and Lys58Glu are amino acid substitutions with which stability against alkali can be especially improved.

The immunoglobulin-binding protein of the present invention may further have at least one amino acid substitution other than these. Examples of the other amino acid substitution(s) include the amino acid substitution Gly29Ala, which is known to increase structural stability (<NPL>). Thus, examples of the modified amino acid sequence also include an amino acid sequence which is the same as an unmodified amino acid sequence exemplified above (such as the amino acid sequence of SEQ ID NO:<NUM>) except that the modified amino acid sequence has the amino acid substitution(s) at the particular position(s) and the other amino acid substitution(s) such as Gly29Ala. Thus, examples of the immunoglobulin-binding protein of the present invention also include a protein containing an amino acid sequence which is the same as an unmodified amino acid sequence exemplified above (such as the amino acid sequence of SEQ ID NO:<NUM>) except that the amino acid sequence of the immunoglobulin-binding protein has the amino acid substitution(s) at the particular position(s) and the other amino acid substitution(s) such as Gly29Ala. In other words, the immunoglobulin-binding protein of the present invention may be, for example, a protein containing the same amino acid sequence as an unmodified amino acid sequence exemplified above (such as the amino acid sequence of SEQ ID NO: <NUM>) except for the presence of the amino acid substitution(s) at the particular position(s) and the other amino acid substitution(s) such as Gly29Ala.

Specific examples of the combination of the amino acid substitutions include the amino acid substitutions Lys7Glu, Asn21Tyr, and Gly29Ala; the amino acid substitutions Lys7Glu, Asn21Tyr, Gly29Ala, and Lys58Glu; the amino acid substitutions Lys4Arg, Lys7Glu, Asn21Tyr, Gly29Ala, and Lys58Glu; the amino acid substitutions Lys4Arg, Lys7Glu, Asn21Tyr, Gly29Ala, Lys49Met, and Lys58Glu; the amino acid substitutions Asn3Ile, Lys4Arg, Lys7Glu, Asn21Tyr, Gly29Ala, and Lys58Glu; the amino acid substitutions Asn3Thr, Lys4Arg, Lys7Glu, Asn21Tyr, Gly29Ala, and Lys58Glu; the amino acid substitutions Lys4Arg, Lys7Glu, Asn11Lys, Asn21Tyr, Gly29Ala, and Lys58Glu; the amino acid substitutions Lys4Arg, Lys7Glu, Asn11Tyr, Asn21Tyr, Gly29Ala, and Lys58Glu; the amino acid substitutions Lys4Arg, Lys7Glu, Asn21Tyr, Gly29Ala, and Lys58Val; the amino acid substitutions Lys4Arg, Lys7Glu, Asn11Lys, Glu15Ala, Asn21Tyr, Gly29Ala, and Lys58Glu; the amino acid substitutions Lys4Arg, Lys7Glu, Asn11Lys, Asn21Tyr, Gly29Ala, and Lys58Gly; the amino acid substitutions Lys4Arg, Lys7Glu, Asn11Lys, Glu15Ala, Asn21Tyr, Gly29Ala, Val40Ala, and Lys58Glu; the amino acid substitutions Asn3Ile, Lys4Arg, Lys7Glu, Asn11Lys, Glu15Ala, Asn21Tyr, Gly29Ala, and Lys58Glu; the amino acid substitutions Lys4Arg, Lys7Glu, Asn11Lys, Glu15Ala, Asn21Tyr, Gly29Ala, and Lys58Asp; and the amino acid substitutions Lys4Arg, Lys7Glu, Asn11Lys, Glu15Ala, Asn21Tyr, Gly29Ala, and Lys58Val.

Thus, the immunoglobulin-binding proteins of the present invention include the following immunoglobulin-binding proteins. These immunoglobulin-binding proteins are preferred from the viewpoint of improving stability against alkali. An immunoglobulin-binding protein containing an amino acid sequence which is the same as the amino acid sequence of SEQ ID NO: <NUM> except that the amino acid sequence of the immunoglobulin-binding protein has the amino acid substitution Asn21Tyr (an immunoglobulin-binding protein containing the amino acid sequence of SEQ ID NO:<NUM>). An immunoglobulin-binding protein containing an amino acid sequence which is the same as the amino acid sequence of SEQ ID NO: <NUM> except that the amino acid sequence of the immunoglobulin-binding protein has the amino acid substitutions Lys7Glu, Asn21Tyr, and Gly29Ala (an immunoglobulin-binding protein containing the amino acid sequence of SEQ ID NO: <NUM>). An immunoglobulin-binding protein containing an amino acid sequence which is the same as the amino acid sequence of SEQ ID NO: <NUM> except that the amino acid sequence of the immunoglobulin-binding protein has the amino acid substitutions Lys7Glu, Asn21Tyr, Gly29Ala, and Lys58Glu (an immunoglobulin-binding protein containing the amino acid sequence of SEQ ID NO: <NUM>). An immunoglobulin-binding protein containing an amino acid sequence which is the same as the amino acid sequence of SEQ ID NO: <NUM> except that the amino acid sequence of the immunoglobulin-binding protein has the amino acid substitutions Lys4Arg, Lys7Glu, Asn21Tyr, Gly29Ala, and Lys58Glu (an immunoglobulin-binding protein containing the amino acid sequence of SEQ ID NO: <NUM>). An immunoglobulin-binding protein containing an amino acid sequence which is the same as the amino acid sequence of SEQ ID NO: <NUM> except that the amino acid sequence of the immunoglobulin-binding protein has the amino acid substitutions Lys4Arg, Lys7Glu, Asn21Tyr, Gly29Ala, Lys49Met, and Lys58Glu (an immunoglobulin-binding protein containing the amino acid sequence of SEQ ID NO:<NUM>). An immunoglobulin-binding protein containing an amino acid sequence which is the same as the amino acid sequence of SEQ ID NO: <NUM> except that the amino acid sequence of the immunoglobulin-binding protein has the amino acid substitutions Asn3Ile, Lys4Arg, Lys7Glu, Asn21Tyr, Gly29Ala, and Lys58Glu (an immunoglobulin-binding protein containing the amino acid sequence of SEQ ID NO:<NUM>). An immunoglobulin-binding protein containing an amino acid sequence which is the same as the amino acid sequence of SEQ ID NO: <NUM> except that the amino acid sequence of the immunoglobulin-binding protein has the amino acid substitutions Asn3Thr, Lys4Arg, Lys7Glu, Asn21Tyr, Gly29Ala, and Lys58Glu (an immunoglobulin-binding protein containing the amino acid sequence of SEQ ID NO:<NUM>). An immunoglobulin-binding protein containing an amino acid sequence which is the same as the amino acid sequence of SEQ ID NO: <NUM> except that the amino acid sequence of the immunoglobulin-binding protein has the amino acid substitutions Lys4Arg, Lys7Glu, Asn11Lys, Asn21Tyr, Gly29Ala, and Lys58Glu (an immunoglobulin-binding protein containing the amino acid sequence of SEQ ID NO:<NUM>). An immunoglobulin-binding protein containing an amino acid sequence which is the same as the amino acid sequence of SEQ ID NO: <NUM> except that the amino acid sequence of the immunoglobulin-binding protein has the amino acid substitutions Lys4Arg, Lys7Glu, Asn11Tyr, Asn21Tyr, Gly29Ala, and Lys58Glu (an immunoglobulin-binding protein containing the amino acid sequence of SEQ ID NO:<NUM>). An immunoglobulin-binding protein containing an amino acid sequence which is the same as the amino acid sequence of SEQ ID NO: <NUM> except that the amino acid sequence of the immunoglobulin-binding protein has the amino acid substitutions Lys4Arg, Lys7Glu, Asn21Tyr, Gly29Ala, and Lys58Val (an immunoglobulin-binding protein containing the amino acid sequence of SEQ ID NO:<NUM>). An immunoglobulin-binding protein containing an amino acid sequence which is the same as the amino acid sequence of SEQ ID NO: <NUM> except that the amino acid sequence of the immunoglobulin-binding protein has the amino acid substitutions Lys4Arg, Lys7Glu, Asn11Lys, Glu15Ala, Asn21Tyr, Gly29Ala, and Lys58Glu (an immunoglobulin-binding protein containing the amino acid sequence of SEQ ID NO:<NUM>). An immunoglobulin-binding protein containing an amino acid sequence which is the same as the amino acid sequence of SEQ ID NO: <NUM> except that the amino acid sequence of the immunoglobulin-binding protein has the amino acid substitutions Lys4Arg, Lys7Glu, Asn11Lys, Asn21Tyr, Gly29Ala, and Lys58Gly (an immunoglobulin-binding protein containing the amino acid sequence of SEQ ID NO:<NUM>). An immunoglobulin-binding protein containing an amino acid sequence which is the same as the amino acid sequence of SEQ ID NO: <NUM> except that the amino acid sequence of the immunoglobulin-binding protein has the amino acid substitutions Lys4Arg, Lys7Glu, Asn11Lys, Glu15Ala, Asn21Tyr, Gly29Ala, Val40Ala, and Lys58Glu (an immunoglobulin-binding protein containing the amino acid sequence of SEQ ID NO:<NUM>). An immunoglobulin-binding protein containing an amino acid sequence which is the same as the amino acid sequence of SEQ ID NO: <NUM> except that the amino acid sequence of the immunoglobulin-binding protein has the amino acid substitutions Asn3Ile, Lys4Arg, Lys7Glu, Asn11Lys, Glu15Ala, Asn21Tyr, Gly29Ala, and Lys58Glu (an immunoglobulin-binding protein containing the amino acid sequence of SEQ ID NO:<NUM>). An immunoglobulin-binding protein containing an amino acid sequence which is the same as the amino acid sequence of SEQ ID NO: <NUM> except that the amino acid sequence of the immunoglobulin-binding protein has the amino acid substitutions Lys4Arg, Lys7Glu, Asn11Lys, Glu15Ala, Asn21Tyr, Gly29Ala, and Lys58Asp (an immunoglobulin-binding protein containing the amino acid sequence of SEQ ID NO:<NUM>). An immunoglobulin-binding protein containing an amino acid sequence which is the same as the amino acid sequence of SEQ ID NO:<NUM> except that the amino acid sequence of the immunoglobulin-binding protein has the amino acid substitutions Lys4Arg, Lys7Glu, Asn11Lys, Glu15Ala, Asn21Tyr, Gly29Ala, and Lys58Val (an immunoglobulin-binding protein containing the amino acid sequence of SEQ ID NO:<NUM>).

"Amino acid at position X in the amino acid sequence of SEQ ID NO: <NUM>" means the amino acid present at the Xth position as counted from the N-terminus of the amino acid sequence of SEQ ID NO: <NUM>. "Amino acid residue corresponding to the amino acid at position X of the amino acid sequence of SEQ ID NO: <NUM>" in a certain amino acid sequence means the amino acid residue, in the certain amino acid sequence, which is placed at the same position as the Xth amino acid in the amino acid sequence of SEQ ID NO: <NUM> in an alignment of the certain amino acid sequence with the amino acid sequence of SEQ ID NO: <NUM>. For example, in a case of the amino acid substitution Asp2Glu, "amino acid residue corresponding to the aspartic acid at position <NUM> of SEQ ID NO: <NUM>" in a certain amino acid sequence means the amino acid residue, in the certain amino acid sequence, which is placed at the same position as the aspartic acid at position <NUM> of the amino acid sequence of SEQ ID NO:<NUM> in an alignment of the certain amino acid sequence with the amino acid sequence of SEQ ID NO: <NUM>. "Amino acid residue corresponding to the Xth amino acid in the amino acid sequence of SEQ ID NO:<NUM>" in the amino acid sequence of SEQ ID NO: <NUM> means the Xth amino acid itself in the amino acid sequence of SEQ ID NO:<NUM>. Thus, the positions of the amino acid substitutions exemplified above (that is, the amino acid substitutions at the particular positions described above) do not necessarily represent the absolute positions in the immunoglobulin-binding protein of the present invention, but represent relative positions based on the amino acid sequence of SEQ ID NO: <NUM>. That is, for example, in cases where the immunoglobulin-binding protein of the present invention contains insertion, deletion, or addition of an amino acid residue(s) in the N-terminal side relative to the positions of the amino acid substitutions exemplified above, the absolute positions of the amino acid substitutions may change
in accordance therewith. The positions of the amino acid substitutions exemplified above in the immunoglobulin-binding protein of the present invention can be specified by, for example, alignment of the amino acid sequence of the immunoglobulin-binding protein of the present invention with the amino acid sequence of SEQ ID NO: <NUM>. The alignment can be carried out by, for example, using an alignment program such as BLAST. The same applies to the positions of the amino acid substitutions exemplified above in any amino acid sequences such as variant sequences of the amino acid sequence of SEQ ID NO: <NUM>. The amino acid residues before the amino acid substitutions exemplified above (that is, the amino acid substitutions at the particular positions described above) represent the types of the unsubstituted amino acid residues in the amino acid sequence of SEQ ID NO:<NUM>, and may or may not be conserved in unmodified amino acid sequences other than the amino acid sequence of SEQ ID NO: <NUM>.

Disclosed but not part of the claimed subject-matter, an immunoglobulin-binding protein may be composed of a modified amino acid sequence, or may further contain another amino acid sequence (such as an oligopeptide). Disclosed but not part of the claimed subject-matter, animmunoglobulin-binding protein may further contain another amino acid sequence, for example, in the N-terminal side or the C-terminal side thereof. In other words, in an immunoglobulin-binding protein, another amino acid sequence may be added to, for example, the N-terminal side or the C-terminal side of the modified amino acid sequence. The other amino acid sequence is not limited as long as it does not deteriorate the immunoglobulin-binding capacity or the stability of the immunoglobulin-binding protein. For example, the type and the length of the other amino acid sequence are not limited as long as they do not deteriorate the immunoglobulin-binding capacity or the stability of the immunoglobulin-binding protein.

Disclosed but not part of the claimed subject-matter, an immunoglobulin-binding protein may contain, for example, part of another immunoglobulin-binding domain as well as the immunoglobulin-binding domain selected. For example, in cases where the immunoglobulin-binding protein contains a modified amino acid sequence of domain C, the immunoglobulin-binding protein may further contain part of the region in the N-terminal side of domain C of protein A (domain E, domain D, domain A, and/or domain B/Z), and/or part of the region in the C-terminal side of domain C of protein A.

Further, disclosed but not part of the claimed subject-matter, an immunoglobulin-binding protein may contain, in its N-terminal side or C-terminal side, an oligopeptide useful for the purpose of specifically detecting or separating a target substance. Examples of such an oligopeptide include polyhistidine and polyarginine.

Further, disclosed but not part of the claimed subject-matter, an immunoglobulin-binding protein may contain, in its N-terminal side or C-terminal side, an oligopeptide useful for immobilizing the immunoglobulin-binding protein of the present invention on a solid phase such as a support for chromatography. Examples of such an oligopeptide include oligopeptides containing lysine or cysteine.

Disclosed but not part of the claimed subject-matter, in cases where the immunoglobulin-binding protein contains the above-described other amino acid sequence, for example, the immunoglobulin-binding protein may be produced in a form already containing the other amino acid sequence, or the above-described other amino acid sequence may be separately produced and added to the protein. In cases where the immunoglobulin-binding protein contains the above-described other amino acid sequence, the immunoglobulin-binding protein can be typically produced by expression from a polynucleotide encoding the entire amino acid sequence of the immunoglobulin-binding protein containing the above-described other amino acid sequence. Disclosed but not part of the claimed subject-matter, for example, a polynucleotide encoding the other amino acid sequence may be linked to a polynucleotide encoding the immunoglobulin-binding protein (which, for example, does not contain the other amino acid sequence) such that the other amino acid sequence is added to the N-terminal side or the C-terminal side of the immunoglobulin-binding protein, and then the immunoglobulin-binding protein of the present invention may be expressed. Further, for example, the other amino acid sequence may be chemically synthesized, and chemically bound to the N-terminal side or the C-terminal side of the immunoglobulin-binding protein (which, for example, does not contain the other amino acid sequence).

The immunoglobulin-binding protein of the present invention can be produced by, for example, expression from a polynucleotide encoding the immunoglobulin-binding protein of the present invention. The polynucleotide encoding the immunoglobulin-binding protein of the present invention is also referred to as "polynucleotide of the present invention". More specifically, the polynucleotide of the present invention may be a polynucleotide containing a nucleotide sequence encoding the immunoglobulin-binding protein of the present invention.

The polynucleotide of the present invention can be obtained by, for example, a chemical synthesis method, or a DNA amplification method such as the PCR method. The DNA amplification method can be carried out using, as a template, a polynucleotide containing a nucleotide sequence to be amplified, such as a nucleotide sequence encoding the immunoglobulin-binding protein of the present invention. Examples of the polynucleotide to be used as the template include genomic DNA of an organism that expresses the immunoglobulin-binding protein of the present invention, cDNA of the immunoglobulin-binding protein of the present invention, and vectors containing the polynucleotide of the present invention. The nucleotide sequence of the polynucleotide of the present invention can be designed by, for example, conversion from the amino acid sequence of the immunoglobulin-binding protein of the present invention. In the conversion from the amino acid sequence to the nucleotide sequence, a standard codon table may be used. The conversion is preferably carried out taking into account the codon usage in the host to be transformed with the polynucleotide of the present invention. For example, in cases where the host is Escherichia coli, the conversion may be carried out while avoiding use of the codons AGA/AGG/CGG/CGA for arginine (Arg), ATA for isoleucine (Ile), CTA for leucine (Leu), GGA for glycine (Gly), and CCC for proline (Pro) since the usage of each of these codons is low (that is, the codons are the so-called rare codons). Analysis of the codon usage is possible by, for example, utilizing a public database (such as the Codon Usage Database provided on the website of Kazusa DNA Research Institute).

The polynucleotide of the present invention can be obtained also by, for example, introducing a mutation(s) to a polynucleotide encoding an immunoglobulin-binding protein not having the amino acid substitution(s) exemplified above (that is, the amino acid substitution(s) at the particular position(s) described above) such that the encoded protein has the amino acid substitution(s) exemplified above. Examples of the immunoglobulin-binding protein not having the amino acid substitution(s) exemplified above include a protein containing an unmodified amino acid sequence exemplified above (such as the amino acid sequence of SEQ ID NO: <NUM>). The polynucleotide encoding an immunoglobulin-binding protein not having the amino acid substitution(s) exemplified above can be obtained by, for example, a chemical synthesis method, or a DNA amplification method such as the PCR method. In cases where the immunoglobulin-binding protein of the present invention has two or more amino acid substitutions, these amino acid substitutions may be introduced, for example, simultaneously or sequentially. For example, a mutation(s) may be introduced to a polynucleotide encoding an immunoglobulin-binding protein having at least one amino acid substitution selected from the amino acid substitutions exemplified above, such that the encoded protein has at least one other amino acid substitution selected from the amino acid substitutions exemplified above. Further, the amino acid residue(s) at a certain position(s) may be modified two or more times.

Disclosed herein but not part of the claimed subject-matter, the mutation(s) to be introduced to the polynucleotide is/are not limited to mutations that cause the amino acid substitutions exemplified above. Any mutation(s) such as a mutation(s) for construction of a variant sequence may be introduced to use the polynucleotide as the polynucleotide of the present invention or as a material for obtaining it.

Examples of the method of the introduction of the mutation(s) to the polynucleotide include the error-prone PCR method. The reaction conditions for the error-prone PCR method are not limited as long as they are conditions under which a desired mutation(s) can be introduced to the polynucleotide. For example, a mutation(s) can be introduced to the polynucleotide by adding, to the PCR reaction liquid, the four kinds of substrate deoxynucleotides (dATP/dTTP/dCTP/dGTP) at different concentrations, and MnCl<NUM> at a concentration of <NUM> to <NUM> (preferably <NUM> to <NUM>), and then carrying out PCR. Examples of the method of introducing the mutation(s) to the polynucleotide, other than the error-prone PCR method, include methods in which an agent that acts as a mutagen is allowed to act on the polynucleotide, or the polynucleotide is irradiated with ultraviolet, to introduce the mutation(s) to the polynucleotide. Examples of the agent that acts as a mutagen include mutagenic agents commonly used by those skilled in the art, such as hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine, nitrous acid, sulfurous acid, and hydrazine. Such methods of introducing the mutation(s) to the polynucleotide can be used not only for the introduction of the amino acid substitution(s) exemplified above, but also for construction of a variant sequence (for example, introduction of substitution, deletion, insertion, and/or addition of an amino acid residue(s), and/or changing of the amino acid sequence within the range of homology described above).

For example, the polynucleotide of the present invention may be obtained at once as the entire sequence, or may be obtained by obtaining its partial sequences and then linking the partial sequences to each other. The above description on the method of obtaining the polynucleotide of the present invention is applicable not only to cases where the entire sequence is obtained at once, but also to cases where its partial sequences are obtained.

More specifically, the immunoglobulin-binding protein of the present invention can be produced, for example, by expression of the immunoglobulin-binding protein of the present invention in a transformant having the polynucleotide of the present invention. A transformant having the polynucleotide of the present invention is also referred to as "transformant of the present invention". The transformant of the present invention can express the immunoglobulin-binding protein of the present invention based on the polynucleotide of the present invention contained therein. Thus, the transformant of the present invention is, in other words, a transformant capable of expressing the immunoglobulin-binding protein of the present invention.

The transformant of the present invention can be obtained by, for example, transforming a host using the polynucleotide of the present invention. Thus, for example, the transformant of the present invention may be a host transformed with the polynucleotide of the present invention, may be a host having the polynucleotide of the present invention, or may be a host capable of expressing the immunoglobulin-binding protein of the present invention. The host is not limited as long as the immunoglobulin-binding protein of the present invention can be expressed in cases where the host is transformed with the polynucleotide of the present invention. Examples of the host include animal cells, insect cells, and microorganisms. Examples of the animal cells include COS cells, CHO cells, Hela cells, NIH3T3 cells, and HEK293 cells. Examples of the insect cells include Sf9 and BTI-TN-5B1-<NUM>. Examples of the microorganisms include yeasts and bacteria. Examples of the yeasts include yeasts belonging to the genus Saccharomyces, such as Saccharomyces cerevisiae; yeasts belonging to the genus Pichia, such as Pichia Pastoris; and yeasts belonging to the genus Schizosaccharomyces, such as Schizosaccharomyces pombe. Examples of the bacteria include bacteria belonging to the genus Escherichia, such as Escherichia coli. Examples of the Escherichia coli include the JM109 strain and the BL21 (DE3) strain. A yeast or Escherichia coli is preferably used as the host from the viewpoint of productivity. Escherichia coli is more preferably used as the host.

The polynucleotide of the present invention may be retained in the transformant of the present invention in a mode allowing its expression. More specifically, the polynucleotide of the present invention may be retained such that it is expressed under the regulation of a promoter that functions in the host. In cases where Escherichia coli is used as the host, examples of the promoter that functions in the host include the trp promoter, tac promoter, trc promoter, lac promoter, T7 promoter, recA promoter, and lpp promoter.

In the transformant of the present invention, the polynucleotide of the present invention may be present, for example, on a vector that self-replicates outside the genomic DNA. Thus, for example, the polynucleotide of the present invention can be introduced to the host as an expression vector containing the polynucleotide of the present invention. Thus, in one mode, the transformant of the present invention may be a transformant having an expression vector containing the polynucleotide of the present invention. The expression vector containing the polynucleotide of the present invention is also referred to as "expression vector of the present invention". The expression vector of the present invention can be obtained by, for example, inserting the polynucleotide of the present invention into an appropriate position of an expression vector. The expression vector is not limited as long as it can be stably present and is capable of replication in the host to be transformed therewith. In cases where Escherichia coli is used as the host, examples of the expression vector include the pET plasmid vector, pUC plasmid vector, and pTrc plasmid vector. The expression vector may contain a selection marker such as an antibiotic resistance gene. The appropriate position described above means a position where the insertion does not destroy regions involved in the replication function, selection marker, and transferability of the expression vector. In the process of inserting the polynucleotide of the present invention into the expression vector, the polynucleotide is preferably inserted in a state where it is linked to a functional polynucleotide such as a promoter required for its expression.

In the transformant of the present invention, the polynucleotide of the present invention may be introduced, for example, in the genomic DNA. The introduction of the polynucleotide of the present invention into the genomic DNA can be carried out by, for example, utilizing a gene transfer method based on homologous recombination. Examples of the gene transfer method based on homologous recombination include a method using linear DNA, such as the Red-driven integration method (<NPL>)); a method using a vector containing a temperature-sensitive origin of replication; a method using a vector not having an origin of replication that functions in the host; and a transduction method using a phage.

The transformation of the host using a polynucleotide such as the expression vector of the present invention can be carried out by, for example, a method commonly used by those skilled in the art. For example, in cases where Escherichia coli is selected as the host, the transformation can be carried out by the competent cell method, heat shock method, electroporation method, or the like. By performing screening by an appropriate method after the transformation, the transformant of the present invention can be obtained.

Detailed information on genetic engineering methods such as expression vectors and promoters available for various microorganisms is described in, for example, "<NPL>)". These methods may be used.

In cases where the transformant of the present invention has the expression vector of the present invention, the expression vector of the present invention can be prepared from the transformant of the present invention. For example, from a culture obtained by culturing the transformant of the present invention, the expression vector of the present invention can be prepared by the alkaline extraction method, or by using a commercially available extraction kit such as the QIAprep Spin Miniprep kit (manufactured by QIAGEN).

By culturing the transformant of the present invention, the immunoglobulin-binding protein of the present invention can be expressed. By culturing the transformant of the present invention, the immunoglobulin-binding protein of the present invention can be expressed, and, by recovering the expressed protein, the immunoglobulin-binding protein of the present invention can be produced. Thus, the present invention provides a method of producing the immunoglobulin-binding protein of the present invention, the method including, for example, the steps of: culturing the transformant of the present invention to allow expression of the immunoglobulin-binding protein of the present invention; and recovering the expressed protein. The medium composition and the culture conditions may be appropriately set depending on conditions such as the type of the host and properties of the immunoglobulin-binding protein of the present invention. For example, the medium composition and the culture conditions may be set such that the host can be grown and can express the immunoglobulin-binding protein of the present invention. Examples of media that can be used therefor include media appropriately containing a carbon source, nitrogen source, inorganic salt, and/or other organic components and/or inorganic components. For example, in cases where the host is Escherichia coli, one preferred example of the medium is LB (Luria-Bertani) medium supplemented with necessary nutrient sources (<NUM>% (w/v) tryptone, <NUM>% (w/v) yeast extract, and <NUM>% (w/v) sodium chloride). For selective growth of the transformant of the present invention based on the presence or absence of the expression vector of the present invention introduced, the culture is preferably carried out with a medium supplemented with an antibiotic corresponding to an antibiotic resistance gene contained in the expression vector. For example, in cases where the expression vector contains a kanamycin resistance gene, the medium may be supplemented with kanamycin. The same applies to cases where the polynucleotide of the present invention is introduced in the genomic DNA. The medium may also contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystatin, thioglycolate, and dithiothreitol. The medium may also contain a reagent, such as glycine, which promotes secretion of protein from the transformant into the culture liquid. For example, in cases where the host is Escherichia coli, glycine is preferably added at not more than <NUM>% (w/v) to the medium. For example, in cases where the host is Escherichia coli, the culture temperature may be generally <NUM> to <NUM>, preferably <NUM> to <NUM>, more preferably about <NUM>. For example, in cases where the host is Escherichia coli, the pH of the medium may be pH <NUM> to pH <NUM>, preferably about pH <NUM>. In cases where the immunoglobulin-binding protein of the present invention is expressed under the regulation of an inducible promoter, the induction is preferably carried out so as to allow favorable expression of the immunoglobulin-binding protein of the present invention. For the induction of the expression, for example, an inducer depending on the type of the promoter may be used. Examples of the inducer may include IPTG (Isopropyl-β-D-thiogalactopyranoside). For example, in cases where the host is Escherichia coli, an appropriate amount of IPTG may be added when the turbidity (absorbance at <NUM>) of the culture liquid becomes about <NUM> to <NUM>, and the culture may then be continued to induce expression of the immunoglobulin-binding protein of the present invention. The IPTG may be added at a concentration of, for example, <NUM> to <NUM>, preferably <NUM> to <NUM>. The induction of the expression such as IPTG induction can be carried out, for example, under conditions well known in the art.

The immunoglobulin-binding protein of the present invention can be recovered by separation from the culture by a method suitable for the mode of its expression. The "culture" means the entire culture liquid obtained by the culturing, or part thereof. The part is not limited as long as it is a part containing the immunoglobulin-binding protein of the present invention. Examples of the part include cultured cells of the transformant of the present invention, and the medium after the culturing (that is, the culture supernatant). For example, in cases where the immunoglobulin-binding protein is accumulated in the culture supernatant, the cells can be separated by a centrifugation operation, and the immunoglobulin-binding protein of the present invention can then be recovered from the resulting culture supernatant. For example, in cases where the immunoglobulin-binding protein is accumulated in the cells (including periplasm), the cells may be separated by a centrifugation operation, and then an enzyme treatment agent, surfactant, or the like may be added thereto to disrupt the cells, followed by recovering the immunoglobulin-binding protein of the present invention from the disruption product. The recovery of the immunoglobulin-binding protein of the present invention from the culture supernatant or the disrupted cells can be carried out by, for example, a known method used for separation and purification of protein. Examples of such a method include ammonium sulfate fractionation, ion-exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration chromatography, and isoelectric precipitation.

The immunoglobulin-binding protein of the present invention can be used for, for example, separation or analysis of immunoglobulin (antibody). The immunoglobulin-binding protein of the present invention can be used by, for example, immobilization on an insoluble support. More specifically, separation or analysis of immunoglobulin (antibody) can be carried out using, for example, an immunoglobulin adsorbent comprising: an insoluble support; and the immunoglobulin-binding protein of the present invention immobilized on the insoluble support. The immunoglobulin adsorbent comprising: an insoluble support; and the immunoglobulin-binding protein of the present invention immobilized on the insoluble support; is also referred to as "immunoglobulin adsorbent of the present invention". The "separation of immunoglobulin" includes not only separation of immunoglobulin from a solution in which impurities are present, but also separation of immunoglobulins from each other based on their structures, properties, activities, and/or the like. The insoluble support is not limited. Examples of the insoluble support include supports using a polysaccharide such as agarose, alginate (alginic acid salt), carrageenan, chitin, cellulose, dextrin, dextran, or starch as a raw material; supports using a synthetic polymer such as polyvinyl alcohol, polymethacrylate, poly(<NUM>-hydroxyethyl methacrylate), or polyurethane as a raw material; and supports using a ceramic such as silica as a raw material. Among these, supports using a polysaccharide as a raw material, and supports using a synthetic polymer as a raw material, are preferred as the insoluble support. Examples of the preferred supports include polymethacrylate gels in which hydroxyl groups are introduced, such as TOYOPEARL (manufactured by Tosoh Corporation); agarose gels such as Sepharose (manufactured by GE Healthcare); and cellulose gels such as Cellufine (manufactured by JNC Corporation). The shape of the insoluble support is not limited. The insoluble support may have, for example, a form which allows packing of a column therewith. The insoluble support may be, for example, a granular matter or a nongranular matter. The insoluble support may be, for example, porous or nonporous.

The immunoglobulin-binding protein of the present invention can be immobilized on an insoluble support by, for example, covalent bonding. More specifically, for example, the immunoglobulin-binding protein of the present invention can be immobilized on an insoluble support by covalently binding the immunoglobulin-binding protein of the present invention to the insoluble support through an active group contained in the insoluble support. Thus, the insoluble support may contain an active group. For example, the insoluble support may contain the active group on the surface thereof. Examples of the active group include an N-Hydroxysuccinimide (NHS)-activated ester group, an epoxy group, a carboxyl group, a maleimide group, a haloacetyl group, a tresyl group, a formyl group, and haloacetamide. As the insoluble support containing an active group, for example, a commercially available insoluble support containing an active group may be used as it is, or an active group may be introduced to an insoluble support. Examples of commercially available insoluble supports containing an active group include TOYOPEARL AF-Epoxy-<NUM>, TOYOPEARL AF-Tresyl-<NUM> (these are manufactured by Tosoh Corporation), HiTrap NHS-activated HP Columns, NHS-activated Sepharose <NUM> Fast Flow, Epoxy-activated Sepharose 6B (these are manufactured by GE Healthcare), and SulfoLink Coupling Resin (manufactured by Thermo Fisher Scientific Inc.

Examples of the method of introducing the active group to the surface of the support include a method in which one of two or more active sites contained in a compound is reacted with a hydroxyl group, epoxy group, carboxyl group, amino group, or the like present on the surface of the support.

Examples of the compound for introduction of an epoxy group to a hydroxyl group or an amino group present on the surface of the support include epichlorohydrin, ethanediol diglycidyl ether, butanediol diglycidyl ether, and hexanediol diglycidyl ether.

Examples of the compound for introduction of a carboxyl group to an epoxy group present on the surface of the support include <NUM>-mercaptoacetic acid, <NUM>-mercaptopropionic acid, <NUM>-mercaptobutyric acid, <NUM>-mercaptobutyric acid, glycine, <NUM>-aminopropionic acid, <NUM>-aminobutyric acid, and <NUM>-aminohexanoic acid.

Examples of the compound for introduction of a maleimide group to a hydroxyl group, epoxy group, carboxyl group, or amino group present on the surface of the support include N-(ε-maleimidocaproic acid)hydrazide, N-(ε-maleimidopropionic acid)hydrazide, <NUM>-(<NUM>-N-maleimidophenyl)acetic acid hydrazide, <NUM>-aminomaleimide, <NUM>-aminomaleimide, <NUM>-aminomaleimide, <NUM>-aminomaleimide, <NUM>-(<NUM>-aminophenyl)maleimide, <NUM>-(<NUM>-aminophenyl)maleimide, <NUM>-(maleimido)phenyl isocyanate, <NUM>-maleimidoacetic acid, <NUM>-maleimidopropionic acid, <NUM>-maleimidobutyric acid, <NUM>-maleimidohexanoic acid, N-(α-maleimidoacetoxy)succinimide ester, (m-maleimidobenzoyl) N-hydroxysuccinimide ester, succinimidyl-<NUM>-(maleimidomethyl)cyclohexane-<NUM>-carbonyl-(<NUM>-aminohexanoic acid), succinimidyl-<NUM>-(maleimidomethyl)cyclohexane-<NUM>-carboxylic acid, (p-maleimidobenzoyl) N-hydroxysuccinimide ester, and (m-maleimidobenzoyl) N-hydroxysuccinimide ester.

Examples of the compound for introduction of a haloacetyl group to a hydroxyl group or amino group present on the surface of the support include chloroacetic acid, bromoacetic acid, iodoacetic acid, chloroacetic chloride, bromoacetic chloride, bromoacetic bromide, chloroacetic anhydride, bromoacetic anhydride, iodoacetic anhydride, <NUM>-(iodoacetamido)acetic acid-N-hydroxysuccinimide ester, <NUM>-(bromoacetamido)propionic acid-N-hydroxysuccinimide ester, and <NUM>-(iodoacetyl)aminobenzoic acid-N-hydroxysuccinimide ester.

Examples of the method of introducing the active group to the surface of the support also include a method in which ω-alkenylalkane glycidyl ether is reacted with a hydroxyl group or amino group present on the surface of the support, and then the ω-alkenyl site is halogenated with a halogenating agent to cause its activation. Examples of the ω-alkenylalkane glycidyl ether include allylglycidyl ether, <NUM>-butenylglycidyl ether, and <NUM>-pentenylglycidyl ether. Examples of the halogenating agent include N-chlorosuccinimide, N-bromosuccinimide, and N-iodosuccinimide.

Examples of the method of introducing the active group to the surface of the support also include a method in which the active group is introduced to a carboxyl group present on the surface of the support using a condensing agent and an additive. Examples of the condensing agent include <NUM>-ethyl-<NUM>-(<NUM>-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbodiamide, and carbonyldiimidazole. Examples of the additive include N-hydroxysuccinimide (NHS), <NUM>-nitrophenol, and <NUM>-hydroxybenztriazole.

The immobilization of the immunoglobulin-binding protein of the present invention on the insoluble support can be carried out, for example, in a buffer. Examples of the buffer include acetate buffer, phosphate buffer, MES (<NUM>-morpholinoethanesulfonic acid) buffer, HEPES (<NUM>-(<NUM>-hydroxyethyl)-<NUM>-piperazineethanesulfonic acid) buffer, Tris buffer, and borate buffer. The reaction temperature for the immobilization may be appropriately set depending on, for example, conditions such as reactivity of the active group and stability of the immunoglobulin-binding protein. The reaction temperature for the immobilization may be, for example, <NUM> to <NUM>, preferably <NUM> to <NUM>.

The immunoglobulin adsorbent of the present invention can be used for, for example, separation of immunoglobulin (antibody) by using a column packed with the immunoglobulin adsorbent. More specifically, for example, a solution containing immunoglobulin is applied to a column packed with the immunoglobulin adsorbent of the present invention to allow adsorption of the immunoglobulin to the adsorbent, and then the immunoglobulin that has adsorbed to the adsorbent is eluted. By this, the immunoglobulin can be separated. Thus, the present invention provides, for example, a method of separating immunoglobulin, the method comprising the steps of: applying a solution containing immunoglobulin to a column packed with the immunoglobulin adsorbent of the present invention, to allow adsorption of the immunoglobulin to the adsorbent; and eluting the immunoglobulin that has adsorbed to the adsorbent. The solution containing immunoglobulin may be applied to the column using, for example, liquid transferring means such as a pump. Application of a liquid to a column is also referred to as "transfer of a liquid to a column". The solvent of the solution containing immunoglobulin may be preliminarily replaced using an appropriate buffer before the application to the column. Before the application of the solution containing immunoglobulin to the column, the column may be equilibrated using an appropriate buffer. By the equilibration of the column, for example, separation of the immunoglobulin with higher purity can be expected. Examples of the buffer used for the solvent replacement or the equilibration include phosphate buffer, acetate buffer, and MES buffer. The buffer may be further supplemented with, for example, an inorganic salt such as <NUM> to <NUM> sodium chloride. The buffer used for the solvent replacement and the buffer used for the equilibration may be either the same or different. In cases where components other than the immunoglobulin, such as impurities, are remaining in the column after passing the solution containing immunoglobulin through the column, such components may be removed from the column before the elution of the immunoglobulin that has adsorbed to the immunoglobulin adsorbent. The components other than the immunoglobulin can be removed from the column by, for example, using an appropriate buffer. To the buffer used for the removal of the components other than the immunoglobulin, for example, the description on the buffer used for the solvent replacement or for the equilibration is applicable. The immunoglobulin that has adsorbed to the immunoglobulin adsorbent can be eluted by, for example, reducing the interaction between the immunoglobulin and the ligand (the immunoglobulin-binding protein of the present invention). Examples of the means for reducing the interaction between the immunoglobulin and the ligand (the immunoglobulin-binding protein of the present invention) include lowering of the pH using a buffer, addition of a counter peptide, increasing of the temperature, and changing of the salt concentration. More specifically, the immunoglobulin that has adsorbed to the immunoglobulin adsorbent can be eluted by, for example, using an appropriate eluent. Examples of the eluent include buffers that are more acidic than the buffers used for the solvent replacement or the equilibration. Examples of such buffers include citrate buffer, glycine-HCl buffer, and acetate buffer. The pH of the eluent may be set within a range in which
the function (for example, the binding capacity to antigen) of the immunoglobulin is not deteriorated.

By carrying out the separation of the immunoglobulin (antibody) in such a manner, a separated immunoglobulin, for example, is obtained. Thus, in one mode, the method of separating immunoglobulin may be a method of producing immunoglobulin, more specifically, a method of producing separated immunoglobulin. The immunoglobulin is obtained as, for example, an eluted fraction containing the immunoglobulin. Thus, a fraction containing the eluted immunoglobulin can be collected. The collection of the fraction can be carried out by, for example, an ordinary method. Examples of the method of collecting the fraction include a method in which the collection container is replaced at certain time or volume intervals, a method in which the collection container is replaced depending on the shape of the chromatogram of the eluent, and a method in which fractions are collected using an automatic fraction collector such as an autosampler. Further, immunoglobulin can be recovered from a fraction containing the immunoglobulin. The recovery of the immunoglobulin from the fraction containing the immunoglobulin can be carried out by, for example, a known method used for separation and purification of protein.

The present invention is described below more concretely by way of Examples.

(<NUM>) From the amino acid sequence of immunoglobulin-binding protein of SEQ ID NO: <NUM>, the nucleotide sequence of SEQ ID NO:<NUM> was designed by conversion using the Escherichia coli-type codons. (<NUM>) After synthesis of the nucleotide sequence designed in (<NUM>), PCR was used to prepare a polynucleotide containing the nucleotide sequence of SEQ ID NO:<NUM>. For the PCR, the synthesized polynucleotide was used as a template DNA, and oligonucleotides having the nucleotide sequence of SEQ ID NO:<NUM> (<NUM>'-TAGCCATGGGCGCGGATAACAAGTTC-<NUM>') or SEQ ID NO:<NUM> (<NUM>'-CTACTCGAGTTTCGGAGCTTGCGCATC-<NUM>') were used as PCR primers, to prepare a reaction liquid having the composition shown in Table <NUM>. The reaction liquid was then subjected to <NUM> cycles of reaction wherein each cycle included a first step at <NUM> for <NUM> seconds, a second step at <NUM> for <NUM> seconds, and a third step at <NUM> for <NUM> seconds.

(<NUM>) The polynucleotide obtained was purified, and digested with the restriction enzymes NcoI and XhoI, followed by ligation into the expression vector pET-28a that had been preliminarily digested with the restriction enzymes NcoI and XhoI. Using the resulting ligation product, the Escherichia coli BL21 (DE3) strain was transformed. (<NUM>) The transformant obtained was cultured in LB medium supplemented with <NUM>µg/mL kanamycin, and then the expression vector pET-SpA was extracted using a QIAprep Spin Miniprep kit (manufactured by QIAGEN). (<NUM>) The polynucleotide encoding the immunoglobulin-binding protein and its vicinity in the extract obtained were subjected to cycle sequencing reaction using a Big Dye Terminator Cycle Sequencing ready Reaction kit (manufactured by Life Science), which is based on the chain terminator method. The nucleotide sequence was analyzed using an ABI Prism <NUM> DNA analyzer (manufactured by Life Science), which is a fully automated DNA sequencer. In the analysis, oligonucleotides having the nucleotide sequence of SEQ ID NO:<NUM> (<NUM>'-TAATACGACTCACTATAGGG-<NUM>') or SEQ ID NO:<NUM> (<NUM>'-TATGCTAGTTATTGCTCAG-<NUM>') were used as sequencing primers.

As a result of the sequence analysis, it could be confirmed that the expression vector pET-SpA contains a polynucleotide having the nucleotide sequence of SEQ ID NO:<NUM> inserted therein.

To the polynucleotide portion (SEQ ID NO:<NUM>) encoding the immunoglobulin-binding protein inserted in the expression vector pET-SpA prepared in Example <NUM>, mutations were randomly introduced by error-prone PCR.

(<NUM>) Using the pET-SpA prepared in Example <NUM> as a template DNA, error-prone PCR was carried out. For the error-prone PCR, a reaction liquid having the composition shown in Table <NUM> was prepared, and the reaction liquid was heat-treated at <NUM> for <NUM> minutes, followed by performing <NUM> cycles of reaction wherein each cycle included a first step at <NUM> for <NUM> seconds, a second step at <NUM> for <NUM> seconds, and a third step at <NUM> for <NUM> seconds, and then finally performing heat treatment at <NUM> for <NUM> minutes. By the error-prone PCR, mutations were well introduced into the polynucleotide (SEQ ID NO:<NUM>) encoding the immunoglobulin-binding protein. The average mutation introduction rate was <NUM>% to <NUM>%.

(<NUM>) The PCR product obtained was purified, and digested with the restriction enzymes NcoI and XhoI, followed by ligation into the expression vector pET-28a that had been preliminarily digested with the restriction enzymes NcoI and XhoI. (<NUM>) After completion of the ligation reaction, the Escherichia coli BL21 (DE3) strain was transformed using the reaction liquid, and then cultured (<NUM>, <NUM> hours) on LB plate medium supplemented with <NUM>µg/mL kanamycin. The resulting colonies, formed on the plate, were provided as a random mutant library.

For the immunoglobulin-binding protein expressed by each selected transformant, the position of the amino acid substitution in terms of the wild-type immunoglobulin-binding protein (having no amino acid substitution) (SEQ ID NO:<NUM>) and the remaining activity [%] after <NUM> hours of alkali treatment at <NUM> using <NUM> NaOH are summarized in Table <NUM> and Table <NUM>. It can be said that immunoglobulin-binding proteins in which at least any one of the amino acid substitutions Asp2Glu (this expression represents the fact that the aspartic acid at position <NUM> of SEQ ID NO:<NUM> is substituted to glutamic acid; the same applies hereinafter), Lys4Arg, Lys49Met, Asn6Asp, Lys7Glu, Asn21Tyr, Lys42Arg, and Lys58Glu occurred from the amino acid sequence of SEQ ID NO: <NUM> have improved alkaline stability compared to the wild-type immunoglobulin-binding protein (SEQ ID NO: <NUM>).

Among the immunoglobulin-binding proteins having the amino acid substitutions shown in Table <NUM> and Table <NUM>, the immunoglobulin-binding protein in which the amino acid substitution Asn21Tyr occurred, which had the highest remaining activity, was named SpA1, and the expression vector containing the polynucleotide encoding SpA1 was named pET-SpA1. The amino acid sequence of SpA1 is shown in SEQ ID NO:<NUM>, and the nucleotide sequence encoding SpA1 is shown in SEQ ID NO: <NUM>.

The methods of the preparation of the five kinds of immunoglobulin-binding proteins shown in (a) to (e) are described below.

The results are shown in Table <NUM>. All of the mutant-type immunoglobulin-binding proteins evaluated herein (SpA2 (SEQ ID NO: <NUM>), SpA3a (SEQ ID NO:<NUM>), SpA4a (SEQ ID NO:<NUM>), SpA5a (SEQ ID NO:<NUM>), and SpA3b (SEQ ID NO:<NUM>)) showed higher remaining activities compared to the wild-type immunoglobulin-binding protein (SEQ ID NO: <NUM>). Thus, these mutant-type immunoglobulin-binding proteins were found to have improved alkaline stability.

Among the mutant-type immunoglobulin-binding proteins evaluated in Example <NUM>, SpA4a (Example <NUM>(c)) was selected, and mutations were randomly introduced to the polynucleotide portion encoding SpA4a (SEQ ID NO: <NUM>) by error-prone PCR.

For the immunoglobulin-binding protein expressed by each transformant selected in (<NUM>), the position of the amino acid substitution in terms of SpA4a (SEQ ID NO: <NUM>) and the remaining activity [%] after <NUM> hours of alkali treatment at <NUM> using <NUM> NaOH are summarized in Table <NUM> and Table <NUM>. It can be said that immunoglobulin-binding proteins in which at least any one of the amino acid substitutions Asn3Ile, Asn3Thr, Asn11Lys, Asn11Tyr, and Lys(Glu)58Val (this expression represents the fact that the lysine at position <NUM> of SEQ ID NO: <NUM> was once substituted to glutamic acid in the preparation of SpA4a, which substitution was further followed by substitution to valine) occurred from the amino acid sequence of SEQ ID NO: <NUM> have improved alkaline stability compared to SpA4a (SEQ ID NO:<NUM>).

The amino acid sequence of the protein in which the amino acid substitution Asn3<NUM>Ile (this expression represents the fact that the aspartic acid at position <NUM> of SEQ ID NO: <NUM> is substituted to isoleucine; the same applies hereinafter) occurred (which was named SpA5b) from the amino acid sequence of SEQ ID NO: <NUM> is shown in SEQ ID NO:<NUM>; the amino acid sequence of the protein in which the amino acid substitution Asn3<NUM>Thr occurred (which was named SpA5c) is shown in SEQ ID NO:<NUM>; the amino acid sequence of the protein in which the amino acid substitution Asn11<NUM>Lys occurred (which was named SpA5d) is shown in SEQ ID NO:<NUM>; the amino acid sequence of the protein in which the amino acid substitution Asn11<NUM>Tyr occurred (which was named SpA5e) is shown in SEQ ID NO:<NUM>; and the amino acid sequence of the protein in which the amino acid substitution Lys(Glu)<NUM><NUM>Val occurred (which was named SpA4b) is shown in SEQ ID NO:<NUM>.

Among the mutant-type immunoglobulin-binding proteins evaluated in Example <NUM>, SpA5d (Example <NUM>) was selected, and mutations were randomly introduced to the polynucleotide portion encoding SpA5d (SEQ ID NO:<NUM>) by error-prone PCR.

For the immunoglobulin-binding protein expressed by each transformant selected in (<NUM>), the position of the amino acid substitution in terms of SpA5d (SEQ ID NO:<NUM>) and the remaining activity [%] after <NUM> hours of alkali treatment at <NUM> using <NUM> NaOH are summarized in Table <NUM>. It can be said that immunoglobulin-binding proteins in which at least any one of the amino acid substitutions Glu15Ala and Lys(Glu)58Gly occurred from the amino acid sequence of SEQ ID NO:<NUM> have improved alkaline stability compared to SpA5d (SEQ ID NO:<NUM>).

The amino acid sequence of the protein in which the amino acid substitution Glu15<NUM>Ala (this expression represents the fact that the glutamic acid at position <NUM> of SEQ ID NO:<NUM> is substituted to alanine; the same applies hereinafter) occurred (which was named SpA6a) from the amino acid sequence of SEQ ID NO:<NUM> is shown in SEQ ID NO:<NUM>; and the amino acid sequence of the protein in which the amino acid substitution Lys(Glu)<NUM><NUM>Gly occurred (which was named SpA5f) is shown in SEQ ID NO:<NUM>.

Among the mutant-type immunoglobulin-binding proteins evaluated in Example <NUM>, SpA6a (Example <NUM>) was selected, and mutations were randomly introduced to the polynucleotide portion encoding SpA6a (SEQ ID NO:<NUM>) by error-prone PCR.

For the immunoglobulin-binding protein expressed by each transformant selected in (<NUM>), the position of the amino acid substitution in terms of SpA6a (SEQ ID NO:<NUM>) and the remaining activity [%] after <NUM> hours of alkali treatment at <NUM> using <NUM> NaOH are summarized in Table <NUM>, Table <NUM> and Table <NUM>. It can be said that immunoglobulin-binding proteins in which at least any one of the amino acid substitutions Val40Ala, Asn3Ile, Lys(Glu)58Asp, and Lys(Glu)58Val occurred from the amino acid sequence of SEQ ID NO:<NUM> have improved alkaline stability compared to SpA6a (SEQ ID NO:<NUM>).

The amino acid sequence of the protein in which the amino acid substitution Val40<NUM>Ala (this expression represents the fact that the valine at position <NUM> of SEQ ID NO:<NUM> is substituted to alanine; the same applies hereinafter) occurred (which was named SpA7a) from the amino acid sequence of SEQ ID NO:<NUM> is shown in SEQ ID NO:<NUM>; the amino acid sequence of the protein in which the amino acid substitution Asn3<NUM>Ile occurred (which was named SpA7b) is shown in SEQ ID NO:<NUM>; the amino acid sequence of the protein in which the amino acid substitution Lys(Glu)<NUM><NUM>Asp occurred (which was named SpA6b) is shown in SEQ ID NO:<NUM>; and the amino acid sequence of the protein in which the amino acid substitution Lys(Glu)<NUM><NUM>Val occurred (which was named SpA6c) is shown in SEQ ID NO:<NUM>.

Claim 1:
An immunoglobulin-binding protein comprising an amino acid sequence which is the same as an amino acid sequence of an immunoglobulin-binding domain of protein A except that the amino acid sequence of the immunoglobulin-binding protein comprises the amino acid sequence of any of SEQ ID NOs: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> to <NUM>, <NUM>, <NUM>, and <NUM> to <NUM>.