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Mutant immunoglobulin kappa chain variable region-binding peptide

An immunoglobulin, binding technology, applied in the direction of immunoglobulin, carrier-bound/immobilized peptide, hybrid peptide, etc., to achieve the effect of excellent selective adsorption capacity

Inactive Publication Date: 2018-05-22
KANEKA CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, compared to the case of SpA, reported examples of mutation introduction in PpL are limited, especially with regard to the acid elution pH of antibodies adsorbed to affinity separation matrices, although for SpA there are multiple Reports that elution is possible at ~4.0 (Non-Patent Document 1, Patent Documents 7-8), but for PpL, there is still room for improvement

Method used

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  • Mutant immunoglobulin kappa chain variable region-binding peptide
  • Mutant immunoglobulin kappa chain variable region-binding peptide
  • Mutant immunoglobulin kappa chain variable region-binding peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0123] Example 1: Preparation of various mutant VL-κ binding peptides of PpL

[0124] (1) Expression plasmid preparation

[0125] From the amino acid sequence of LB5t-Wild.1d (SEQ ID NO: 16), the base sequence (SEQ ID NO: 22) encoding the peptide was designed by reverse translation. It should be noted that, based on experimental conditions, the nucleotide sequence was designed to encode an amino acid sequence with Glu-Gln added to the N-terminus and Gly added to the C-terminus. The 1-2 residue addition sequence comes from the B5 domain of wild-type PpL. Then, the preparation method of the expression plasmid is shown in figure 2 . The DNA encoding LB5t-Wild.1d was prepared by ligating two double-stranded DNAs (f1 and f2) having the same restriction enzyme site, and introduced into the multiple cloning site of the expression vector. Actually, preparation of coding DNA and introduction into the vector were carried out simultaneously by ligating two kinds of double-stranded D...

Embodiment 2

[0135] Example 2: Affinity evaluation of various LB5t mutants to aRSV-Fab

[0136] (1) Preparation of Fab fragments from IgG

[0137]As a raw material of humanized monoclonal IgG preparation, it is prepared by fragmenting it into Fab fragment and Fc fragment with papain, and separating and purifying only the Fab fragment. Specifically, an anti-RSV monoclonal IgG (generic name "Palivizumab", product name "Synagis", AbbVie company) preparation whose light chain is formed from a κ chain was dissolved in a buffer for papain digestion ( 0.1M AcOH-AcONa, 2mM EDTA, 1mM cysteine, pH5.5), add "PapainAgarose from papaya latex" papain-immobilized agarose (SIGMA company), and mix it with a rotator, while at 37 ° C Incubate for about 8 hours. From the reaction solution (mixed with Fab fragments and Fc fragments) separated from papain-immobilized agarose, it was recovered in the flow-through fraction by affinity chromatography using a MabSelect SuRe column (GE Healthcare Bioscience). IgG...

Embodiment 3

[0143] Example 3: Evaluation of the aRSV-Fab acid dissociation pH of various LB5t mutants

[0144] (1) Preparation of Fab fragment immobilized carrier

[0145] An affinity separation matrix immobilized with the aRSV-Fab obtained in Example 2 was produced using a commercially available coupling column for ligand immobilization in which the coupling target functional group was amino groups.

[0146] As the water-insoluble substrate, 1 mL of a commercially available prepacked column ("Hitrap NHS activated HP" manufactured by GE Healthcare Bioscience) was used. The column uses cross-linked agarose as the matrix, and has introduced active groups for the immobilization of protein ligands whose coupling target functional groups are amino groups. Therefore, the ligands were immobilized according to the product instructions. An operation of passing 1 mM HCl cooled in an ice bath through 2 mL at a flow rate of 1 mL / min was performed three times to remove isopropanol in the column.

[...

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Abstract

The purpose of the present invention is to provide: a mutant immunoglobulin kappa chain variable region-binding peptide characterized by the acid dissociation pH of an immunoglobulin kappa chain variable region-containing protein being shifted to the neutral side compared to prior to the introduction of a mutation; an affinity separation matrix comprising the peptide as a ligand; and a method forproducing a kappa chain variable region-containing protein using the affinity separation matrix. Another purpose of the present invention is to provide DNA that encodes the peptide, a vector containing the DNA, and a transformant that is transformed by the vector. The problem addressed by the present invention is solved by using a peptide into which a mutation has been introduced at an appropriatesite as an affinity ligand for a kappa chain variable region binding domain of protein L derived from a Peptostreptococcus magnus strain.

Description

technical field [0001] The present invention relates to an immunoglobulin κ chain variable region-binding peptide that has improved the acid dissociation properties of the κ chain variable region of an immunoglobulin, an affinity separation matrix having the peptide as a ligand, and an affinity separation matrix using the A method for producing a protein comprising an immunoglobulin κ chain variable region from an isolation matrix, DNA encoding the peptide, a vector containing the DNA, and a transformant transformed with the vector. Background technique [0002] One of the important functions of proteins is the function of specifically binding to a specific molecule. This function plays an important role in the immune response and signal transduction in the living body. Technological development of applying this action to the separation and purification of useful substances has been widely carried out. As an example of practical industrial application, a protein A affinity...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C07K1/22C07K14/195C07K19/00C12N1/15C12N1/19C12N1/21C12N5/10G01N33/53
CPCC07K1/22C07K19/00C12N5/10C12N15/09G01N33/53C07K14/315C07K14/195C07K16/00C07K16/1027C07K17/00C07K2317/55
Inventor 吉田慎一
Owner KANEKA CORP
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