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Staphylococcus protein A as well as purification preparation method and application thereof

A staphylococcal protein and protein technology, which is applied to the preparation method of peptides, biochemical equipment and methods, immunoglobulin, etc., can solve the problems of insufficient purity and IgG purity, and achieve simple preparation process, high purification efficiency, and durable The effect of high alkalinity

Active Publication Date: 2021-10-19
赣江中药创新中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Protein A of this structure binds a large amount of IgG, while its non-Fc binding domain can bind some miscellaneous proteins, resulting in insufficient purity of the eluted IgG. Generally, the purity of industrially synthesized antibodies can exceed 90% after one-step protein A affinity chromatography. %
But for monoclonal antibodies, this purity is often not enough

Method used

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  • Staphylococcus protein A as well as purification preparation method and application thereof
  • Staphylococcus protein A as well as purification preparation method and application thereof
  • Staphylococcus protein A as well as purification preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0030] A staphylococcal protein A, the amino acid sequence of the B functional domain of the staphylococcal protein A is shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0031] A method for the purification and preparation of staphylococcal protein A, the method comprising

[0032] The B functional domain of the ProteinA protein derived from Staphylococcus was partially mutated to form ProteinA-1 and ProteinA-2;

[0033] The recombinant plasmid is constructed, the prokaryotic system induces expression, and the expressed recombinant Protein A protein is separated and purified by Ni affinity chromatography and ion exchange chromatography, and the purity is greater than 95%.

[0034] In this embodiment, the proteinA-1 mutation site includes: A176V, N178H, N181D, Q184A, E190K, N198T, N203G, G204A, N218A, A221S, D228E, A229S total 12 mutations, the amino acid sequence is as shown in SEQ ID NO : 1.

[0035] In this example, the proteinA-2 mutation sites include: A176V, Q184H, E190K, N198...

Embodiment 2

[0041] 1.1 Protein A protein gene synthesis modification design:

[0042] The mutation sites include: A176V, N178H, N181D, Q184A, E190K, N198T, N203G, G204A, N218A, A221S, D228E, A229S, a total of 12 mutated ProteinA-1 proteins, and A176V, Q184H, E190K, N198S, N203G, G204A, D211H, N218A, A221S, D228E, A229S total 11 mutations of Protein A-2 protein, the purpose is to enhance the stability and alkali resistance of the target protein.

[0043] 1.2 ProteinA recombinant plasmid construction:

[0044] A protein purification preparation method and application of the present invention, according to the gene synthesis transformation design in 1.1, entrust GenScript to synthesize the corresponding gene, and connect it into the pET30a (GenScript) vector to construct a recombinant plasmid. In order to better illustrate the superior performance of the ProteinA of the present invention, the wild-type ProteinA (WT) was also commissioned to be synthesized by a company as a negative control. ...

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Abstract

The invention discloses staphylococcus protein A as well as a purification preparation method and application thereof. The method comprises the following steps: connecting a protein Protein A gene modified by genetic engineering into an expression vector to construct a recombinant plasmid; introducing the recombinant plasmid into a prokaryotic host cell for expression and carrying out Ni affinity and ion-exchange chromatography purification, thereby obtaining the target protein with the purity greater than 95%. The purification preparation method has the characteristics of simple purification process, high yield and the like. Meanwhile, the prepared Protein A has good immunoglobulin IgG binding force, alkali resistance and thermal stability. And the affinity medium prepared by coupling the solid phase carrier with Protein A has the advantages of good IgG adsorption effect, high sample loading amount and alkali resistance. The Protein A protein prepared by the invention and a medium prepared by coupling the Protein A protein can play an important role in the field of monoclonal antibody purification and immunodiagnosis research.

Description

technical field [0001] The invention relates to the technical field of cloning and transforming Protein A in genetic engineering, in particular to a staphylococcal protein A, a purification preparation method and an application thereof. Background technique [0002] In the middle of the 20th century, Jensen discovered a protein that could widely bind to human and rabbit serum antibodies. This protein was first called Protein A in 1964. Protein A is a cell wall protein of Staphylococcus aureus with a molecular weight of 42KDa. It can specifically bind to the Fc region of various immunoglobulins and weakly bind to the Fab region or light chain. Natural Protein A has 5 IgG binding domains E, D, A, B, C and a non-Fc binding domain of unknown function. The 5 different domains can bind the Fc fragment of IgG with strong specific affinity. A ProteinA molecule can bind at least two IgG molecules. At the same time, IgA, IgM or IgE may also combine with ProteinA. Protein A of this ...

Claims

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Application Information

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IPC IPC(8): C07K14/31C12N15/70C07K1/18C07K16/00C07K1/22G01N33/53B01J20/281C12R1/19
CPCC07K14/31C12N15/70C07K16/00C07K1/22G01N33/53B01J20/281Y02A50/30
Inventor 刘龙英叶贤龙郭志谋于伟高岩华熊京京徐思梦胡文峰
Owner 赣江中药创新中心
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