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Separation and purification method of recombinant proteins A

A recombinant protein, separation and purification technology, applied in the field of bioengineering, can solve problems such as low product purity, and achieve the effects of reducing production costs, increasing dynamic capacity, and improving dynamic capacity.

Inactive Publication Date: 2010-12-22
DALIAN UNIV OF TECH
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Problems solved by technology

However, this method needs to add a specific enzyme to remove the histidine tag during the purification process, and the product obtained by this method has low purity
[0014] In summary, although the affinity chromatography method using IgG as ligand has some defects, it is still higher than other methods in terms of the purity of the product obtained in a single pass.

Method used

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  • Separation and purification method of recombinant proteins A
  • Separation and purification method of recombinant proteins A
  • Separation and purification method of recombinant proteins A

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Preparation of Fc fragment

[0029] (1) Papain enzymatically digests IgG

[0030] Dissolve an appropriate amount of papain in 10 mM PBS buffer solution with pH 7.4 for a certain period of time, and filter. Add a certain amount of activator cysteine ​​and chelating agent EDTA to make the final concentrations reach 0.02mol / L and 0.01mol / L respectively, add IgG to keep the concentration at 0.5-10mg / ml, and adjust the pH after adding reactants When the value is between 6-7, react in a water bath at 40°C for 3-6h. Finally, the terminator iodoacetamide was added to make the concentration reach 0.03mol / L.

[0031] (2) Purify the Fc fragment using recombinant protein A affinity chromatography column

[0032] Pack the affinity chromatography filler with recombinant protein A as the ligand, equilibrate the column with 5 column volumes of equilibration buffer (10mM PBS, pH 7.4), and detect the absorbance at 280nm until the baseline is balanced. Slowly pass the enzymolysis solu...

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Abstract

The invention relates to a separation and purification method of recombinant proteins A, which comprises the steps of: preparing batch IgG Fc segments; directionally immobilizing the Fc segments; and separating and purifying the recombinant proteins A through an affinity chromatography technology by using the Fc segments as aglycones. The method belongs to the technical field of biological engineering. The purification of the proteins A finally obtained and detected by SAS-PAGE and HPLC is more than 95 percent. The method has the advantages of lower cost, high efficiency and simple and convenient operation, and not only can be used for purifying the proteins A in a laboratory scale way, but also can be used for producing industrialized proteins A.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a method for purifying recombinant protein A by using the Fc fragment of IgG as an affinity chromatography technology. Background technique [0002] Staphylococcus aureus protein A (Staphylococcus aureus protein A, SPA) is a cell wall-associated protein of Staphylococcus aureus, which is a single-chain polypeptide structure and has the ability to specifically bind to the Fc segment of immunoglobulin molecules in the blood of humans and other mammals , mainly adsorb IgG and IgG-containing immune complexes. It is precisely because of the specific binding ability of protein A to various immunoglobulins of humans and other mammals that it is widely used as an immunological reagent (coupled with various reporter molecules for histochemistry, Western, ELISA, etc. Antibody detection); connected to a solid-phase carrier for the separation and purification of antibodies; clinically ...

Claims

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Application Information

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IPC IPC(8): C07K14/31C07K1/22
Inventor 贾凌云侯率林雪张嘉玉任军谢键
Owner DALIAN UNIV OF TECH
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