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Fixed-point immobilization method for recombinant protein A affinity ligands

A fixed-point and affinity technology, applied in chemical instruments and methods, separation methods, carrier binding/immobilization of peptides, etc., can solve the problems of destroying disulfide bonds, affecting the effect of fixed-point immobilization, and high production cost of protein ligands. The effect of improving adsorption efficiency, reducing selling price and reducing production cost

Active Publication Date: 2015-02-18
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to aim at the high cost of production of protein ligands, the key raw material existing in the existing affinity chromatography fillers that use proteins as ligands, and the reducing environment used for fixed-point immobilization of terminal sulfhydryl groups will destroy the disulfide bonds of proteins themselves, and at the same time Its own sulfhydryl group will also participate in the reaction, which will affect the fixed-point immobilization effect and other common problems. It provides a method for the fixed-point immobilization of protein affinity ligands. This method does not need to purify the recombinant protein, and directly fixes the protein from the crude material solution. Ligand

Method used

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  • Fixed-point immobilization method for recombinant protein A affinity ligands
  • Fixed-point immobilization method for recombinant protein A affinity ligands
  • Fixed-point immobilization method for recombinant protein A affinity ligands

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1. Preparation of protein A modified by site-directed formhylation

[0030] 1. Vector construction and transformation screening:

[0031] Protein A gene sequence: The signal peptide and membrane binding domain are removed from the full-length gene of protein A, and the gene sequences of five antibody Fc fragment binding domains A, B, C, D, and E are used as the gene sequence of protein A. The full length is 888bp, as shown in SEQ ID NO.2.

[0032] Through two rounds of PCR amplification, insert the specific sequence recognized by FGE shown in SEQ ID NO.1 into the 5' end of the protein A gene sequence, the specific method is:

[0033] (1) The names and sequences of the primers used are shown in Table 1, and the underlined part is the enzyme cutting site.

[0034] Table 1. Primer names and their sequences

[0035]

[0036] (2)PCR

[0037] The first round of PCR: the protein A gene sequence was ligated into the pET23a plasmid through the Nde Ⅰ and BamH Ⅰ res...

Embodiment 2

[0046] Example 2. Preparation of surface hydrazide functionalized agarose gel microspheres:

[0047] The preparation method of the solid phase chromatographic matrix with active hydrazide groups includes the activation of epichlorohydrin, the addition of diamino reagents for epoxy ring opening, further addition of dialdehyde reagents to form a chromatographic matrix with active aldehyde groups, and finally the use of bisamino reagents to form a chromatographic matrix with active aldehyde groups. The hydrazide reagent reacts with the aldehyde group on the chromatographic matrix to form a solid phase chromatographic matrix with active hydrazide groups. Wherein the diaminopropyl imine is preferred as the diamino reagent, glutaraldehyde is preferred as the dialdehyde reagent, and adipate dihydrazide is preferred as the dihydrazide reagent. The specific method is as follows:

[0048] (1) Epichlorohydrin activation: Take 10mL of Sepharose CL-4B, wash it with single distilled water ...

Embodiment 3

[0052] Example 3. Direct site-specific immobilization of aldehyde protein A

[0053] Take 4mL of the hydrazide functionalized agarose gel in Example 2, divide it into four equal parts, each 1mL, add 1mL of the cell crushing supernatant in Example 1 respectively, in order to investigate the reaction of different catalysts on hydrazide and aldehyde groups 100mM aniline, 10mM p-phenylenediamine, and 5mM 3,5-diaminobenzoic acid were added to the reaction system as catalysts, and PBS was added as a control. After reacting at 37°C for 12 hours, the solid-phase matrix was collected by centrifugation, and the solid-phase matrix was collected with 1M The solid-phase matrix was washed 3 times with PBS of NaCl to wash away non-specifically bound proteins. The chromatographic matrix of site-fixed protein A was obtained and stored in PBS solution containing 0.02% sodium azide at 4°C. Further, 200mM hydroxylamine hydrochloride was used to cut off the hydrazide and aldehyde group under the ...

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Abstract

The invention provides a fixed-point immobilization method for recombinant protein A affinity ligands. According to the method, an FGE (formylglycine generating enzyme) identification gene sequence is inserted at the tail end of a gene sequence of coding target protein with a genetic engineering means, an expression vector of the target protein is constructed, then the target protein and FGE are co-expressed in escherichia coli, and intracellular aldehyde catalytic conversion of the target protein is realized; then fixed-point immobilization is realized through selective reactions of hydrazide groups on the surface of a medium and aldehyde groups on the surface of the protein. With the adoption of the method, the target recombinant protein can be directly fixed in a broken supernatant of a cell, conditions are mild, steps are simple, the immobilization time is greatly shortened, and the immobilization cost is greatly reduced.

Description

technical field [0001] The invention belongs to the field of protein immobilization, and relates to a method for fixed-point immobilization of protein affinity ligands, in particular to a method for fixed-point immobilization of recombinant protein A affinity ligands. Background technique [0002] In recent years, monoclonal antibody drugs have developed rapidly. From 1986 when the US FDA approved the first antibody drug on the market to 2013, 38 antibody drugs have been successfully launched. Currently, monoclonal antibody drugs account for 8 of the 20 biopharmaceuticals with sales exceeding US$2 billion, showing an increasing trend year by year. In addition to the monoclonal antibody drugs already on the market, there are also a large number of monoclonal antibody drugs under clinical research. These monoclonal antibody drug treatments mainly involve malignant tumors, autoimmune diseases such as rheumatoid arthritis, and rejection after organ transplantation. Because mono...

Claims

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Application Information

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IPC IPC(8): C07K17/08C07K17/04B01D15/20
Inventor 贾凌云臧柏林任军徐丽
Owner DALIAN UNIV OF TECH
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