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

一种免疫球蛋白、蛋白质的技术，应用在对免疫球蛋白具有亲和性的蛋白质和免疫球蛋白结合性亲和配体领域，能够解决没有导入等问题，达到高耐碱性、充分抗体酸解离特性的效果

Inactive Publication Date: 2012-02-29
KANEKA CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the mutation of substituting Gly at position 29 with Ala was disclosed in 1987 (Non-Patent Document 4), there has been no introduction of mutations replacing amino acids other than Ala until now.

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0138] Example 1: Preparation of DNA encoding wild-type C domain (C-wild type)

[0139]Using the expression vector pNK3262NX encoding wild-type protein A as a template, the oligonucleotide primers of sequence number 19-20 were used for PCR to amplify the DNA fragment (177bp) encoding C-wild type (sequence number 5). The pNK3262NX used as a template is a protein A expression vector in which a part of pNK3262 has been modified, which encodes wild-type protein A from which a part of the cell wall binding domain X and the like have been removed (International Publication No. WO 06 / 004067). The nucleotide sequence encoding C-wild type obtained in this Example is shown in SEQ ID NO: 21. In addition, the oligonucleotide primers shown in SEQ ID NO: 19-20 were designed such that the DNA amplified using them had restriction enzyme recognition sites of BamHI and EcoRI on the outside of the gene encoding C-wild type point, and has a Cys residue on the C-terminal side (after Lys-58) of ...

Embodiment 2

[0142] Example 2: Preparation of DNA encoding wild type B domain (B-wild type)

[0143] Such as figure 1 As shown, the amino acid sequence of B-wild type (SEQ ID NO: 4) is a sequence obtained by introducing mutations of T23N, V40Q, K42A, E43N, and I44L into C-wild type. Therefore, the DNA encoding the B-wild type was prepared by introducing mutations T23N, V40Q, K42A, E43N, and I44L into the DNA encoding the C-wild type (SEQ ID NO: 21). Using the C-wild-type expression plasmid obtained in Example 1 as a template, and using the oligonucleotide primers shown in SEQ ID NO: 22-23, such a plasmid was obtained, which contained the encoding method and introduced into the C-wild-type by the QuickChange method. The gene for the amino acid sequence of the T23N mutation. In addition, using the obtained plasmid as a template, using the oligonucleotide primers shown in SEQ ID NO: 24-25, to obtain such a GST fusion type B-wild type expression plasmid, which contains V40Q, K42A introduc...

Embodiment 3

[0146] Example 3: Introduction of mutations into Gly-29

[0147] Using the C-wild-type expression plasmid obtained in Example 1 and the B-wild-type expression plasmid obtained in Example 2 as templates, using the primers of sequence numbers 27-52 shown in Table 1, various coding variants were obtained using the QuickChange method. Gene.

[0148] Using the C-wild-type expression plasmid as a template, replace Gly-29 in the amino acid sequence of SEQ ID NO: 5 with Val, Leu, Ile, Tyr, Phe, Thr, Trp, Ser, Asp, Glu, Arg, His or Met, Various expression plasmids encoding the C domain variant (C-G29X) described in any one of SEQ ID NO:6-18 were obtained. Similarly, using the B-wild-type expression plasmid as a template, replace Gly-29 in the amino acid sequence of SEQ ID NO: 4 with Val, Arg, Asp or Trp to obtain the B structure described in any one of the coding sequence numbers 53-56 Various expression plasmids for the domain variant (B-G29X).

[0149] Transformation of Escheric...

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Abstract

Disclosed are: a novel modified protein A ligand having a superior property of causing the acid dissociation of antibodies compared to those of existing modified protein A ligands; and a novel modified protein A ligand having high alkali resistance. Specifically disclosed are: a protein having an affinity for an immunoglobulin, which is characterized by comprising an amino acid sequence produced by substituting at least one Gly residue by an amino acid residue other than an Ala residue in an amino acid sequence derived from any one of E-domain, D-domain, A-domain, B-domain and C-domain of protein A, and which is also characterized by having a reduced affinity for an Fab region contained in the immunoglobulin compared to a protein comprising an amino acid sequence produced by substituting the Gly residue by an Ala residue in the amino acid sequence derived from any one of E-domain, D-domain, A-domain, B-domain and C-domain of protein A; and a protein having an affinity for an immunoglobulin, which is characterized by having improved chemical stability under alkaline conditions compared to a corresponding domain.

Description

technical field [0001] The present invention relates to a protein specifically bound to an antibody, an affinity separation matrix using the protein as an immunoglobulin-binding affinity ligand, and a method for using the matrix to separate and purify or to remove antibodies by adsorption. technical background [0002] Antibodies have the function of specifically binding to substances called antigens, and the function of detoxifying and removing antigen-bearing factors in cooperation with other biomolecules or cells. The name of the so-called antibody is a name that emphasizes such a function of binding to an antigen, and it can be called an "immunoglobulin" as a substance. [0003] In recent years, along with the development of genetic engineering, protein engineering, and cell engineering, the development of pharmaceutical products that utilize antibody functions—called antibody drugs—has become increasingly popular. Compared with traditional drugs, antibody drugs are exp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09B01J20/281C07K1/22C07K14/31C12N1/15C12N1/19C12N1/21C12N5/10C12P21/02
CPCB01D15/3809B01J20/286B01J20/3244B01J20/3274C07K14/31C07K14/47C07K2319/00C07K2319/23C07K2319/24
Inventor 吉田慎一村田大高野昌行赤木隼也井口惠太中野喜之
Owner KANEKA CORP
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