Protein capable of binding specifically to immunoglobulin, and immunoglobulin-binding affinity ligand
View Patent ↗An object of the present invention is to create a novel engineered Protein A ligand having better antibody dissociation properties in the acidic condition compared with known engineered Protein A ligands. The present invention provides a protein having an affinity for an immunoglobulin, including an amino acid sequence obtained by introducing, into an amino acid sequence derived from any of E, D, A, B and C domains of Protein A, at least one amino acid substitution at any one or more of amino acid residues corresponding to positions 31 to 37 of the A, B and C domains (positions 29 to 35 of the E domain, positions 34 to 40 of the D domain), which are conserved in all the domains, the protein having a lower affinity for an Fab region of an immunoglobulin than a protein having the amino acid sequence before introduction of the substitution.
1. A protein having an affinity for an immunoglobulin, comprising the amino acid sequence having substitutions and a sequence identity of not less than 90% to the amino acid sequence of SEQ ID NO:5, wherein the amino acid residue corresponding to position 29 of SEQ ID NO:5 is Ala, wherein the amino acid residue corresponding to position 33 of SEQ ID NO:5 is Glu, and/or the amino acid residue corresponding to position 36 of SEQ ID NO:5 is Arg, and wherein the protein has a lower affinity for an Fab region of an immunoglobulin than the protein before introduction of the substitutions.
2. A multi-domain protein, comprising two or more proteins according to claim 1 , connected together.
3. The multi-domain protein according to claim 2 ,
wherein the proteins connected together are different from one another.
4. The multi-domain protein according to claim 2 ,
wherein the number of the proteins connected together is 2 to 5.
5. An affinity separation matrix, comprising:
the protein according to claim 1 as an affinity ligand, and a carrier made of a water-insoluble base material on which the protein is immobilized.
6. The affinity separation matrix according to claim 5 ,
wherein the water-insoluble base material comprises a synthetic polymer or a polysaccharide.
7. The affinity separation matrix according to claim 6 ,
wherein the polysaccharide is cellulose.
8. An affinity separation matrix, comprising the protein according to claim 1 as an affinity ligand, and a carrier made of a water-insoluble base material on which the protein is immobilized, wherein the affinity separation matrix binds to a protein containing an Fc region of an immunoglobulin.
9. The affinity separation matrix according to claim 8 ,
wherein the protein containing an Fc region of an immunoglobulin is an immunoglobulin G or an immunoglobulin G derivative,
wherein the immunoglobulin G derivative is at least one member selected from the group consisting of chimeric immunoglobulin Gs, humanized immunoglobulin Gs, and immunoglobulin G whose Fc region has a modified sugar chain and which has a function as an immunoglobulin G and is bound by Protein A.
10. A protein having an affinity for an immunoglobulin, comprising an amino acid sequence having substitutions and a sequence identity of not less than 90% to the amino acid sequence of SEQ ID NO:5, wherein the amino acid residue corresponding to position 29 of SEQ ID NO:5 is Ala, wherein the amino acid residue corresponding to position 33 of SEQ ID NO:5 is Glu or Thr, and/or the amino acid residue corresponding to position 36 of SEQ ID NO:5 is Arg or Ile, and wherein the protein has a lower affinity for an Fab region of an immunoglobulin than the protein before introduction of the substitutions.
11. A DNA encoding the protein according to claim 1 .
12. A vector comprising the DNA according to claim 11 .
13. A transformant which is obtainable by transformation of a host with the vector according to claim 12 .
14. The transformant according to claim 13 ,
wherein the host is a gram-positive bacterium.
15. The transformant according to claim 14 ,
wherein the gram-positive bacterium is a bacterium of Brevibacillus.
16. The transformant according to claim 15 ,
wherein the bacterium of Brevibacillus is Brevibacillus choshinensis.
17. A method for producing the protein according to claim 1 , the method comprising utilizing a transformant which is obtainable by transformation of a host with a vector comprising a DNA encoding the protein, or a cell-free protein synthesis system using a DNA encoding the protein.
18. The production method according to claim 17 , comprising:
accumulating the protein intracellularly and/or in a periplasmic space of the transformant; and/or
secreting the protein extracellularly from the transformant.