Immunoglobulin binding protein, and affinity support using same

An immunoglobulin and protein technology, applied in the field of immunoglobulin-binding proteins, can solve the problems of difficult antibody purification and low antibody purification efficiency, and achieve high binding activity and high antibody dissociation rate

Pending Publication Date: 2020-05-05
JSR CORPORATIOON +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

On the other hand, under mild acidic conditions, the antibody is not easily eluted from the column, therefore, the purification efficiency of the antibody is reduced

Method used

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  • Immunoglobulin binding protein, and affinity support using same
  • Immunoglobulin binding protein, and affinity support using same

Examples

Experimental program
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Effect test

Embodiment 1

[0271] Example 1 Production of Immunoglobulin κ Chain Binding Proteins (KBP1-14)

[0272] Acquired from the artificial gene synthesis manufacturer. The gene encoding the protein comprising a plurality of immunoglobulin binding domain variants (variant domains) composed of any one of the amino acid sequences shown in SEQ ID NO: 10 to 23 was respectively encoded into pET- A plasmid derived from the 24a(+) vector. These plasmids were transformed into Escherichia coli competent cells BL21(DE3) (manufactured by NEWENGLAND BioLabs) to obtain transformed cells.

[0273]The resulting transformed cells were incubated at 37°C until the absorbance (OD600) reached about 10. Then, IPTG (manufactured by Sigma-Aldrich) was added to a final concentration of 1 mM, and further incubated at 37° C. for 4 hours to express the recombinant immunoglobulin κ chain-binding protein. Cells were recovered and disrupted in Tris buffer at pH 9.5. The recombinant immunoglobulin-binding protein was purifie...

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Abstract

A protein L-derived immunoglobulin binding protein with an improved antibody dissociation rate under acidic conditions, and an affinity support using the same are provided. This immunoglobulin bindingprotein contains at least one mutant of an immunoglobulin binding domain, and this affinity support includes a solid-phase support to which said immunoglobulin binding protein is bonded. The mutant of the immunoglobulin binding domain is formed from an amino acid sequence which is at least 85% identical with an amino acid sequence represented in any of sequence numbers 1-9 and which has a given mutation, and said mutant has immunoglobulin k-chain binding activity.

Description

technical field [0001] The present invention relates to an immunoglobulin-binding protein, an affinity carrier using the same, and a method for isolating an antibody or fragment thereof using the affinity carrier. Background technique [0002] In recent years, antibodies have been widely used as research reagents, antibody medicines, and the like. Antibodies for these reagents and pharmaceuticals are usually purified by affinity chromatography. Affinity purification of antibodies generally uses a column immobilized with a ligand that is a substance that specifically binds to immunoglobulin. As the ligand, immunoglobulin-binding proteins such as protein A, protein G, and protein L are generally used. [0003] In the affinity purification of antibodies, generally speaking, after the antibody is adsorbed on the column under neutral conditions, impurities are removed by washing, and then the column is exposed to acidic conditions to dissociate the adsorbed antibody from the li...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/195B01D15/38B01J20/24B01J20/30C07K1/22C12N1/15C12N1/19C12N1/21C12N5/10C12N15/31C12N15/63C12P21/02
CPCC07K1/22C07K14/195C12N5/10C12N15/63C12P21/02B01D15/3804B01D15/3809B01J20/24B01J20/288B01J20/3085B01J2220/52G01N33/54366
Inventor 安冈润一吉村贵一芳贺智亮
Owner JSR CORPORATIOON
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