US 10,065,995 B2 · Protein for affinity-separation matrix

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.

Claims13

Dependent claims

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.

1 more element

Dependencies are inferred from the claim text.

Protein for affinity-separation matrix

Life of this patent

Protein for affinity-separation matrix

Life of this patent

Abstract

Claims13