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Preparation method of recombinant Protein A protein and affinity chromatography medium

A protein and histidine technology, applied in the field of affinity chromatography, to achieve the effects of good alkaline stability, improved alkaline stability, and excellent IgG binding ability

Active Publication Date: 2022-04-12
PINGHU YOUPU BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no relevant published literature that proposes site-directed mutagenesis of glutamine in the E domain of Protein A to histidine, lysine, leucine, serine, threonine or arginine

Method used

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  • Preparation method of recombinant Protein A protein and affinity chromatography medium
  • Preparation method of recombinant Protein A protein and affinity chromatography medium
  • Preparation method of recombinant Protein A protein and affinity chromatography medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] 1. Protein mutagenesis of Protein A protein with mutations in B domain or Z domain

[0108] In order to improve the chemical stability of Protein A protein, there are more and more studies on modifying it by protein engineering to prepare recombinant Protein A protein as a ligand to improve the stability of affinity chromatography column alkali. Patent CN104804073B introduces mutations in the protein A protein amino acid sequence into leucine, arginine and lysine, and the recombinant protein A protein shows an enhanced binding ability to IgG compared with the natural protein A protein. In patent US6399750 B1, cysteine ​​is mutated into Protein A protein, and the obtained recombinant Protein A protein is easy to immobilize on the surface of water-insoluble carrier, which has the advantages of high binding capacity and the ligand is not easy to fall off. Patent CN102365361A mutated into Protein A protein tyrosine and histidine, and found that compared with before mutation...

Embodiment 2

[0159] Example 2 Recombinant Protein A protein with D domain mutation

[0160] 1. Optimization of Protein A protein gene

[0161] Based on the technical scheme of the invention, based on the sequence of SEQ ID NO: 21, the asparagine at the 6th position of the amino acid sequence of the specified D domain has been mutated to be selected from glutamine, serine, histidine, lysine, arginine amino acid, leucine, tyrosine; the asparagine at the 9th position has been mutated to be selected from aspartic acid, glutamine, histidine, lysine; the glutamine at the 12th position has been mutated Mutated to be selected from lysine, leucine, serine, threonine, arginine; glutamic acid at position 18 has been mutated to be selected from histidine, lysine, leucine, serine, threonine Amino acid, arginine, the designed single-point amino acid mutation sequence is shown in SEQ ID NO:22~SEQ ID NO:43; at the same time, in a preferred embodiment, two site mutations are performed on the sequence o...

Embodiment 3

[0195] Example 3 Recombinant Protein A protein with A domain mutation

[0196] 1. Optimization of Protein A protein gene

[0197] The A domain sequence is defined by SEQ ID NO:64

[0198] Based on the technical scheme of the invention, amino acid sequences such as SEQ ID NO:65~SEQ ID NO:86 are designed and translated into nucleotide sequences and entrusted to a gene synthesis company for gene synthesis.

[0199] 2. Expression and purification of recombinant Protein A protein with A domain mutation

[0200] The synthesized amino acid sequence was transformed into Escherichia coli BL21(DE3) through the recombinant plasmid PET30a vector, and the fermentation culture was carried out through LB liquid medium. After fermentation, the substances in the periplasm are dispersed into the culture medium by thermal cracking and centrifuged to obtain the expression substance solution.

[0201] Purify the separated expression substance solution through the IgG chromatography medium, l...

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PUM

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Abstract

The invention discloses a recombinant Protein A protein, the recombinant Protein A protein is composed of one or more mutants of an E structural domain, a D structural domain, an A structural domain and a B structural domain or a Z structural domain of the Protein A protein, and in addition, the recombinant Protein A protein can also be composed of a repeated unit formed by combining 4-8 repeated mutants, on the other hand, the invention discloses an affinity chromatography medium containing a repetitive unit of a recombinant Protein A protein mutant or a mutant combination, and a preparation method and application of the affinity chromatography medium. Based on the recombinant Protein A protein provided by the invention, the recombinant Protein A protein has better chemical stability and alkali resistance and more excellent IgG binding capacity, so that the recombinant Protein A protein provided by the invention is used as an affinity ligand for preparing an affinity chromatography filler and is applied to separation and purification of antibodies.

Description

technical field [0001] The invention relates to the field of affinity chromatography, in particular to a method for preparing recombinant Protein A protein and an affinity chromatography medium. Background technique [0002] In recent years, the field of biopharmaceuticals has been booming, and the global biopharmaceutical market is expanding day by day. As the largest category of therapeutic biological agents, antibody drugs have more obvious curative effect and lower toxicity than traditional therapies in cancer treatment. Antibody drugs include monoclonal antibodies (naked monoclonal antibodies), bispecific antibodies and antibody-drug conjugates (conjugated monoclonal antibodies). Major pharmaceutical companies focus on maximizing the productivity of their respective antibody drug production processes while controlling related costs, so the purification process is crucial in the preparation of antibody drugs. [0003] At present, the existing purification technology ma...

Claims

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Application Information

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IPC IPC(8): C07K14/31C07K16/00C07K1/22B01D15/38
Inventor 张洪石海涛
Owner PINGHU YOUPU BIOTECH CO LTD
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