Protein having affinity for immunoglobulin, and immunoglobulin-binding affinity ligand

Patent metadata

Field	Value
Publication number	US-9403883-B2
Application number	US-201013258655-A
Country	US
Kind code	B2
Filing date	Mar 24, 2010
Priority date	Mar 24, 2009
Publication date	Aug 2, 2016
Grant date	Aug 2, 2016

Abstract

An object of the present invention is to create a novel engineered Protein A ligand having better antibody dissociation properties in the presence of an acid than conventional engineered Protein A ligands and a further object of the present invention is to create a novel engineered Protein A ligand having higher alkali resistance. The present invention is to provide a protein having an affinity for an immunoglobulin, including an amino acid sequence derived from any of E, D, A, B and C domains of Protein A, wherein at least one Gly residue in the amino acid sequence is replaced with an amino acid other than Ala, and the protein has a lower affinity for an Fab region of an immunoglobulin than a protein including an amino acid sequence in which the Gly residue is replaced with Ala. Also, the present invention is to provide the protein having an affinity for an immunoglobulin, which has improved chemical stability in an alkaline condition compared to the corresponding domain.

First claim

The invention claimed is:

1. A protein having an affinity for an immunoglobulin, comprising an amino acid sequence derived from,
C domain of protein A of SEQ ID NO: 5,

wherein a glycine (Gly) residue corresponding to position 29 of the C domain, which is conserved in the C domain of Protein A, is replaced with an amino acid other than alanine (Ala), and
the protein has a lower affinity for a Fab region of an immunoglobulin than a protein comprising an amino acid sequence in which the Gly residue is replaced with Ala,
wherein the amino acid other than Ala is any of leucine (Leu), isoleucine (Ile), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), glutamic acid (Glu), arginine (Arg), and methionine (Met), and which has 85% or higher sequence identity to the amino acid sequence of SEQ ID NO: 5.

2. The protein according to claim 1 ,
wherein the Gly residues corresponding to position 29 of the C domain is the Gly residue at position 29 of the C domain.

3. The protein according to claim 1 ,
wherein the amino acid sequence before introduction of the mutation is an amino acid sequence of SEQ ID NO: 5.

4. The protein according to claim 1 ,
which has improved chemical stability in an alkaline condition compared to a protein having an amino acid sequence before introduction of the mutation.

5. The protein according to claim 1 ,
wherein the amino acid sequence after introduction of the mutation is any of amino acid sequences of SEQ ID NOs: 6 to 18.

6. A multi-domain protein,
wherein two or more of the proteins according to claim 1 are connected together.

7. A multi-domain protein,
wherein two or more different proteins selected from the proteins according to claim 1 are connected together.

8. The multi-domain protein according to claim 6 ,
wherein the number of the domains is 2 to 5.

9. An affinity separation matrix, comprising the protein according to claim 1 as affinity ligand,
wherein the protein is immobilized on a carrier made of a water-insoluble base material.

10. The affinity separation matrix according to claim 9 , which binds to a protein containing an Fc region of an immunoglobulin.

11. The affinity separation matrix according to claim 10 ,
wherein the protein containing an Fc region of an immunoglobulin is an antibody, antibody derivative, antibody fragment, or antibody fragment derivative.

12. The affinity separation matrix according to claim 11 ,
wherein the antibody, antibody derivative, antibody fragment, or antibody fragment derivative is an IgG or an IgG derivative.

13. An affinity separation matrix, comprising the multi-domain protein according to claim 6 as an affinity ligand,
wherein the protein is immobilized on a carrier made of a water-insoluble base material.

14. A DNA encoding the protein according claim 1 .

15. A DNA encoding the multi-domain protein according to claim 6 .

16. A vector comprising the DNA according to claim 14 .

17. A transformant which is obtainable by transformation of a host with the vector according to claim 16 .

18. The transformant according to claim 17 ,
wherein the host is a gram-positive bacterium.

19. The transformant according to claim 18 ,
wherein the gram-positive bacterium is a bacterium of Brevibacillus .

20. The transformant according to claim 19 ,
wherein the bacterium of Brevibacillus is Brevibacillus choshinensis .

21. The DNA according to claim 15 ,
wherein nucleotide sequences encoding the domains have 90 % or lower sequence identity to one another.

22. A method for producing the protein according to claim 1 , the method comprising:
utilizing the transformant which is obtainable by transformation of a host with a vector comprising DNA encoding the protein according to claim 1 .

23. The production method according to claim 22 , further comprising:
accumulating the protein intracellularly or in a periplasmic space of the transformant; and/or
extracellularly secreting the protein from the transformant.

24. A method for separating a protein containing an Fc region of an immunoglobulin, comprising adsorbing the protein containing the Fc region of the immunoglobulin on the affinity separation matrix according to claim 9 .

25. A method for producing the protein according claim 1 , the method comprising:
utilizing a cell-free protein synthesis system using a DNA encoding the protein according to claim 1 .

26. A method for producing the multi-domain protein according to claim 6 , the method comprising:
utilizing a transformant which is obtainable by transformation of a host with a vector comprising DNA encoding the multi-domain protein according to claim 6 .

27. A method for producing the multi-domain protein according to claim 6 , the method comprising:
utilizing a cell-free protein synthesis system using a DNA encoding the protein according to claim 6 .

Assignees

• Yoshida Shinichi
• Murata Dai
• Takano Masayuki
• Akagi Junya
• Iguchi Keita
• Nakano Yoshiyuki
• Kaneka Corp

Inventors

• Yoshida Shinichi
• Murata Dai
• Takano Masayuki
• Akagi Junya
• Iguchi Keita
• Nakano Yoshiyuki

Classifications

• C07K14/31

  from Staphylococcus (G) · CPC title

• C07K2319/24

  containing a MBP (maltose binding protein)-tag · CPC title

• B01D15/3809

  of the antigen-antibody type, e.g. protein A, G or L chromatography · CPC title

• B01J20/3244

  Non-macromolecular compounds · CPC title

• B01J20/3274

  Proteins, nucleic acids, polysaccharides, antibodies or antigens · CPC title