Immunoglobulin-binding protein, and affinity carrier using same

A technology of immunoglobulin and protein, which is applied in the field of immunoglobulin-binding protein, and can solve problems such as low alkali resistance and reduced antibody binding

Pending Publication Date: 2021-04-09
JSR CORPORATIOON +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the native immunoglobulin-binding domain of protein A has low alkali resistance, the affinity purification column immobilized with the native domain will significantly reduce the antibody binding ability during repeated use

Method used

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  • Immunoglobulin-binding protein, and affinity carrier using same
  • Immunoglobulin-binding protein, and affinity carrier using same
  • Immunoglobulin-binding protein, and affinity carrier using same

Examples

Experimental program
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Embodiment

[0322] Hereinafter, an Example is given and this invention is demonstrated more concretely. In addition, the following description generally shows the aspect of this invention, and unless otherwise indicated, this invention is not limited to these descriptions.

reference example 1

[0323] Reference Example 1: Synthesis of Porous Particles

[0324] (1) Add 3.58 g of polyvinyl alcohol (PVA-217 manufactured by Kuraray Co., Ltd.) to 360 g of pure water, heat and stir to dissolve the polyvinyl alcohol, and after cooling, add sodium lauryl sulfate (manufactured by Wako Pure Chemical Industries) 0.36 g, 0.36 g of sodium sulfate (manufactured by Wako Pure Chemical Industries) and 0.18 g of sodium nitrite (manufactured by Wako Pure Chemical Industries) were stirred to prepare an aqueous solution S.

[0325] (2) Dissolve a monomer composition composed of 12.00 g of glycidyl methacrylate (manufactured by Mitsubishi Chemical Corporation) and 1.33 g of divinylbenzene (manufactured by Nippon Steel Chemical Co., Ltd.) in diisobutyl ketone (Mitsui Chemicals Co., Ltd. Chemical Co., Ltd.) 24.43 g to prepare a monomer solution.

[0326] (3) Put the whole amount of the aqueous solution S obtained in (1) into a separable flask, install a thermometer, a stirring blade and a ...

Embodiment 1

[0328] Example 1: Production of immunoglobulin-binding proteins (PrAt-0-32)

[0329] Immunoglobulin-binding proteins PrAt-0-32 were obtained. PrAt-0 is a homotetramer in which an A1V / G29A variant (alkali-resistant variant; non-patent document 1) comprising the C domain of protein A (SEQ ID NO. 3) is connected in series by peptide bonds Immunoglobulin binding protein. PrAt-1 to 32 are variants obtained by introducing the mutations described in Table 1 into the respective immunoglobulin-binding domains of PrAt-0.

[0330] [Table 1]

[0331]

[0332]Expression and purification of PrAt-0 to 32 were carried out as follows. Escherichia coli BL21(DE3) (manufactured by New England Biolabs) was transformed with plasmids encoding PrAt-0 to 32, and the obtained transformants were cultured in a nutrient-rich medium at 37° C. until logarithmic growth phase. Then, the target protein was expressed by adding isopropyl-β-thiogalactopyranoside (manufactured by Wako Pure Chemical Industri...

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Abstract

Provided is an affinity carrier having improved alkali resistance. This immunoglobulin-binding protein contains a mutant polypeptide chain. This affinity carrier includes a solid-phase carrier to which the immunoglobulin-binding protein is bound. The mutant polypeptide chain has an amino acid sequence having at least 85% identity to an amino acid sequence indicated by any of SEQ ID NO: 1-6 and 57-62 and having a predetermined mutation, and has immunoglobulin-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 using the affinity carrier. Background technique [0002] Antibodies have been widely used in research reagents, antibody medicines, and the like in recent years. Antibodies for these reagents and medicines are generally produced through purification by affinity chromatography. In the affinity purification of antibodies, a column immobilized with a ligand, which is a substance that specifically binds to immunoglobulin, is used. As the ligand, immunoglobulin-binding proteins such as protein A are generally used . [0003] Protein A is a cell wall protein from the Gram-positive bacterium Staphylococcus aureus. Protein A has five immunoglobulin-binding domains called E domain, D domain, A domain, B domain, and C domain, and each domain can independently bind to immunoglobulin. Along with the native immunoglobul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C07K1/22C07K14/315C07K16/00C07K17/00C12N1/15C12N1/19C12N1/21C12N5/10C12N15/10C12N15/63C12P21/02G01N33/53G01N33/566
CPCC07K1/22C07K17/00B01D15/3809B01J20/286C07K14/31B01J20/3274C07K16/00
Inventor 芳贺智亮市居敬小野木俊介中村聪本田俊成上出伦敬
Owner JSR CORPORATIOON
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