Protein for affinity-separation matrix
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.
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 .