Protein for affinity-separation matrix

Patent metadata

Field	Value
Publication number	US-10065995-B2
Application number	US-201214007225-A
Country	US
Kind code	B2
Filing date	Mar 26, 2012
Priority date	Mar 25, 2011
Publication date	Sep 4, 2018
Grant date	Sep 4, 2018

Abstract

An object of the present invention is to provide a technique to create novel engineered protein ligands that, when immobilized through a lysine residue (its side chain ε-amino group) which allows for efficient immobilization to a carrier, show the optimum binding capacity and binding efficiency to a target molecule. The present invention provides an engineered protein having a sequence obtained by replacing all the lysine residues in Protein A, which is the most typical protein ligand, with other amino acids, and adding lysine at a terminal; and an affinity separation matrix in which such an engineered protein is immobilized on a water-insoluble carrier by reductive amination or the like. This affinity separation matrix is characterized by its high binding capacity to a target molecule even when the immobilized amount of the ligand is small.

First claim

The invention claimed is:

1. A protein comprising an amino acid sequence obtained by introducing amino acid substitutions for all Lys (lysine) residues, and adding Lys to a terminus, into the amino acid sequence derived from the C domain of protein A having the amino acid sequence of SEQ ID No:5,
wherein not less than half of the amino acid substitutions for all the Lys residues are substitutions of Arg,
wherein 90% or more of Gln-9, Gln-10, Phe-13, Tyr-14, Leu-17, Pro-20, Asn-21, Leu-22, Gln-26, Arg-27, Phe-30, Ile-31, Leu-34, Pro-38, Ser-39, Leu-45, Leu-51, Asn-52, Gln-55, and Pro-57 are conserved in the protein wherein the residue numbers correspond to the residue numbers of the C domain of SEQ ID No:5,
the protein has at least 80% sequence identity to the full length amino acid sequence of SEQ ID No:5, and
wherein the protein is immobilized on a carrier of an affinity separation matrix through the Lys added to the terminus.

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

3. The protein according to claim 1 , wherein all the substitutions are substitutions of Arg.

4. The protein according to claim 2 , wherein two or more of the domains of the Protein A are linked to one another through a linker.

5. The protein according to claim 4 , wherein the linker comprises Lys.

6. An affinity separation matrix comprising: a 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.

7. The affinity separation matrix according to claim 6 , which binds to a protein comprising an Fc region of an immunoglobulin.

8. A DNA encoding a protein according to claim 1 .

9. A vector comprising a DNA according to claim 8 .

10. A transformant obtainable by transforming a vector according to claim 9 into a host cell.

11. A method for producing a protein according to claim 1 which comprises:
using either a cell-free protein synthesis system incorporating a DNA encoding said protein according to claim 9 or a transformant according to claim 10 .

12. A method for preparing an affinity separation matrix, the method comprising:
immobilizing a protein according to claim 1 on a carrier made of a water-insoluble material.

13. A method for purifying a protein containing an Fc region of an immunoglobulin, the method comprising:
adsorbing a protein containing an Fc region of an immunoglobulin to an affinity separation matrix according to claim 6 .

Assignees

• Yoshida Shinichi
• Murata Dai
• Taira Shunichi
• Kaneka Corp

Inventors

• Yoshida Shinichi
• Murata Dai
• Taira Shunichi

Classifications

• C07K2317/92

  Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value · CPC title

• C07K2317/21

  from primates, e.g. man · CPC title

• B01D15/3809

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

• B01J2220/4856

  Proteins, DNA · CPC title

• C07K16/00

  Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies · CPC title