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Support for affinity chromatography and method for isolating immunoglobulin

一种免疫球蛋白、载体的技术，应用在亲和色谱用载体和分离免疫球蛋白领域，能够解决容量降低、碱性条件不稳定等问题

Inactive Publication Date: 2013-08-28
JSR CORPORATIOON +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For a carrier for affinity chromatography using a protein as a ligand, such alkaline conditions are harsh, and the capacity may decrease due to the instability of the ligand.
In particular, various supports for affinity chromatography whose ligands are polypeptides such as protein A are unstable to harsh alkaline conditions

Method used

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  • Support for affinity chromatography and method for isolating immunoglobulin
  • Support for affinity chromatography and method for isolating immunoglobulin
  • Support for affinity chromatography and method for isolating immunoglobulin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0093] 2. Example

[0094] Hereinafter, the filler for affinity chromatography according to the present embodiment will be described more concretely with reference to examples. In addition, the following description generally shows the form of this invention, and there is no particular reason, and this invention is not limited to the said description.

manufacture example 1

[0095] 2.1. Production Example 1 (Synthesis of Porous Particles)

[0096] Dissolve 8.2 g of glycidyl methacrylate (manufactured by MITSUBISHI RAYON), 65.9 g of trimethylolpropane trimethacrylate (manufactured by Sartomer), and 90.6 g of glyceryl monomethacrylate (manufactured by NOF Corporation) Add 2,2'-azobisisobutyronitrile (manufactured by Wako Pure Chemical Industries, Ltd.) to 245.8 g of 2-octanone (manufactured by Toyo Gosei Kogyo Co., Ltd.) and 62 g of acetophenone (manufactured by Wako Pure Chemical Industries, Ltd.) , to prepare an organic monomer solution.

[0097] Next, 8.5 g of polyvinyl alcohol (PVA-217 manufactured by Kuraray), 0.43 g of sodium lauryl sulfate (Emal10G manufactured by Kao Corporation), and 21.3 g of sodium sulfate (manufactured by Wako Pure Chemical Industries, Ltd.) were added to 4240 g of pure water. g, stirred overnight to prepare an aqueous solution.

[0098] Next, the obtained aqueous solution was poured into a 7L separable flask, a thermome...

manufacture example 2

[0100] 2.2. Production example 2 (production of recombinant immunoglobulin-binding protein (JX01))

[0101] In this production example and the following production examples, GE Healthcare's PCR purification kit (PCR Purification Kit) was used for purification of DNA. Restriction enzymes were purchased from New England Biolabs. Enzymes for PCR were purchased from Fermentas. Primers for PCR were purchased from Sigma. Ligation was performed using T4 DNA ligase (ligase) (Invitrogen). DH5α cells were purchased from Invitrogen. BL21 cells were purchased from STRATAGENE. pET24a-d(+) were purchased from Novagen. Unless otherwise specified, all experiments were performed under the conditions recommended by the Supplier.

[0102] 2.2.1. Construction of JX01 plasmid (JX01-pETM11)

[0103] Plasmid JX01-pETM11 containing DNA (SEQ ID NO: 3) encoding the C* domain of the A1V G29A variant, which is the C domain of protein A, was constructed according to the following procedure.

[010...

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PUM

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Abstract

Provided are a support for affinity chromatography, which has excellent alkali resistance, and a method for isolating immunoglobulin. The support for affinity chromatography is a support, onto which a protein ligand represented by general formula (1): R-R2 has been immobilized. In general formula (1), R represents a polypeptide comprising 4 to 30 amino acid residues and containing an amino acid sequence represented by ATK or ASK, and R2 represents a polypeptide comprising 50 to 500 amino acid residues and containing an immunoglobulin-binding domain comprising an amino acid sequence represented by SEQ ID NO: 1 or 2, a partial sequence thereof, or an amino acid sequence having at least 70% homology therewith. (The end of R2 binding to R is the C- or N-terminus of the immunoglobulin-binding domain.)

Description

technical field [0001] The invention relates to a carrier for affinity chromatography and a method for separating immunoglobulins. In particular, it relates to a carrier for affinity chromatography to which a specific ligand is bound, useful for purification of immunoglobulins, and a method for isolating immunoglobulins. Background technique [0002] Affinity chromatography is a chromatography method in which a substance (ligand) that specifically binds to a substance for separation and purification is immobilized on an insoluble carrier, and a column filled with the obtained ligand-immobilized carrier is used. For example, the separation and purification of organism-related substances such as proteins and nucleic acids (Japanese Patent Application Laid-Open No. 6-281638). [0003] As the carrier for affinity chromatography, for example, cross-linked particles of sugar chains represented by agarose gel and particles mainly composed of synthetic polymers can be used. [000...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/22C07K16/00C07K17/08C12N15/09
CPCC07K16/065C07K17/08B01J20/28069B01J20/288B01J20/321B01J20/3217B01J20/3274B01J20/3278B01J20/3293B01J20/28004B01J20/28057B01J20/28078B01D15/3804C07K1/22C07K16/00
Inventor 中村聪福田哲夫冈野友亮盐谷知范田守功二大谷勇李顺成李星考特尼·沃斯
Owner JSR CORPORATIOON
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