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Method for separating and purifying recombinant protein A

A technology for separation, purification and recombinant protein, applied in the field of purification of recombinant protein A, can solve the problems of high cost, low protein recovery rate, low recovery rate, etc., and achieve the effect of low cost and high protein recovery rate

Active Publication Date: 2014-09-24
GUANGZHOU KONCEN BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a protein A separation and purification method with low cost, high recovery rate and suitable for industrial production, so as to solve the problem of low recovery rate or high cost in the current protein A purification method, and at the same time solve the problem of activated carbon adsorption The problem of low protein recovery in the process

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  • Method for separating and purifying recombinant protein A
  • Method for separating and purifying recombinant protein A

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1: Endotoxin removal by activated carbon adsorption

[0040] 1) Take 700g of bacteria containing recombinant protein A, add 2800 mL of PBS buffer (containing 8.0mM disodium hydrogen phosphate, 2.0mM potassium dihydrogen phosphate, 131mM NaCl) according to the mass:volume ratio of 1:4, and stir well Dissolve the bacteria, then add lysozyme to a concentration of 1 mg / ml, stir in a water bath at 60°C for 1 hour, then place in a boiling water bath and stir for 30 minutes, and centrifuge after cooling to obtain a broken supernatant;

[0041] 2) Divide the crushed supernatant into 7 equal parts, add NaCl to each part until the concentration increases by 0.3mol / L, add Triton X-100 to the concentrations of 0, 0.25%, 0.5%, 1%, 2%, 4%, 6%, respectively % (v / v), after stirring evenly, add 4% activated carbon according to the mass:volume ratio, put it in a 60°C water bath and stir for 4 hours for the first adsorption, and centrifuge to obtain the supernatant solution;

[0...

Embodiment 2

[0045] Embodiment 2: Removal of endotoxin by hydrophobic chromatography

[0046] 1) Take 100g of bacteria containing recombinant protein A, add 400mL of PBS buffer (containing

[0047] 8.0mM disodium hydrogen phosphate, 2.0mM potassium dihydrogen phosphate, 131mM NaCl), stir well to dissolve the bacteria, then add lysozyme to a concentration of 1mg / ml, stir in a water bath at 60°C for 1h, then place in a boiling water bath and stir for 30min , after cooling, centrifuge to obtain broken supernatant;

[0048] 2) Add NaCl to the crushed supernatant until the concentration increases to 0.3mol / L, add Triton X-100 to a concentration of 0.5% (v / v), stir evenly, add 4% activated carbon according to the mass:volume ratio, and place in a 60°C water bath Stir for 4 hours for the first adsorption, and centrifuge to obtain a supernatant solution;

[0049] 3) Then add NaCl until the concentration increases by 0.3mol / L, add Triton X-100 until the concentration increases by 0.5% (v / v), sti...

Embodiment 3

[0053] Embodiment 3: Removal of endotoxin by hydrophobic chromatography

[0054] 1) Take 100g of bacteria containing recombinant protein A, add 400mL of PBS buffer (containing

[0055] 8.0mM disodium hydrogen phosphate, 2.0mM potassium dihydrogen phosphate, 131mM NaCl), stir well to dissolve the bacteria, then add lysozyme to a concentration of 1mg / ml, stir in a water bath at 60°C for 1h, then place in a boiling water bath and stir for 30min , after cooling, centrifuge to obtain broken supernatant;

[0056] 2) Add NaCl to the crushed supernatant to increase the concentration to 0.3mol / L, add Triton X-100 to a concentration of 2% (v / v), stir evenly, add 4% activated carbon according to the mass:volume ratio, and place in a 60°C water bath Stir for 4 hours for the first adsorption, and centrifuge to obtain a supernatant solution;

[0057] 3) Then add NaCl until the concentration increases by 0.3mol / L, add Triton X-100 until the concentration increases by 2% (v / v), stir evenly,...

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Abstract

The invention relates to a method for separating and purifying a recombinant protein A, belongs to a protein separation and purification technology in the technical field of bioengineering and provides a method for separating and purifying a recombinant protein A by combining heating treatment and activated carbon adsorption. The purity of the recombinant protein A obtained by the method through separation and purification can be up to 95% or above, the final protein A recovery rate can be up to 60% or above, the endotoxin content in the recovered protein solution is less than 10 EU / mg, and the endotoxin content can be reduced to 1 EU / mg or below if hydrophobic chromatography is further included in the purification method. The method has the advantages of low cost and high recovery rate, is convenient to realize industrial production and provides a feasible route for the large-scale production of the recombinant protein A.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for purifying recombinant protein A and removing endotoxin. Background technique [0002] Staphylococcus aureus Protein A (SPA) is a component of the cell wall of Staphylococcus aureus. SPA can specifically bind to the Fc fragments in human and various mammalian serum IgG molecules, so it has been widely used in the isolation and purification of antibodies, as well as in the clinical treatment of antibody-related diseases. [0003] Due to the wide range of uses of protein A, the market demand is increasing day by day. The direct extraction of natural protein from Staphylococcus aureus not only cannot meet the market demand but also the method of obtaining protein A is highly complicated; Staphylococcus aureus is a pathogenic bacteria, large-scale production is dangerous, and it is prone to produce therapeutic substances A series of problems such as r...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/31C07K1/14C07K1/20
Inventor 陈校园余波光张海珍周彤
Owner GUANGZHOU KONCEN BIOSCI
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