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Method for removing endotoxin in recombinant protein A solution

A recombinant protein and endotoxin technology, applied in the field of bioengineering, can solve the problems of high cost, low recovery rate of recombinant protein A, and poor endotoxin removal effect

Inactive Publication Date: 2017-02-15
湖北中创医疗用品有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In summary, the current methods for removing endotoxin in recombinant protein A solution mainly have the problems of poor endotoxin removal effect, low recovery rate of recombinant protein A, and high cost.

Method used

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  • Method for removing endotoxin in recombinant protein A solution

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Embodiment 1

[0060] A method for removing endotoxin in recombinant protein A solution, comprising the following steps:

[0061] (1) Take 10 g of cells containing recombinant protein A, add PBS buffer and mix the cells to obtain a cell suspension containing recombinant protein A, heat and stir in a water bath at 80° C. for 0.5 h to fully break the cells, the Recombinant protein A has a hexahistidine tag.

[0062] The PBS buffer solution is a mixed solution containing 10 mmol / L disodium hydrogen phosphate, 2 mmol / L sodium dihydrogen phosphate, and 150 mmol / L sodium chloride, and the pH value is adjusted by NaOH to 8;

[0063] The weight ratio of the thalline containing recombinant protein A to PBS buffer is 1:10;

[0064] (2) Add polyethyleneimine, after mixing evenly, centrifuge at 12000r / min, take the supernatant to obtain the supernatant containing recombinant protein A, the weight of the bacterial suspension containing recombinant protein A and polyethyleneimine The ratio is 10000:3; ...

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Abstract

The invention provides a method for removing endotoxin in a recombinant protein A solution. The method is good in endotoxin removal effect, high in the recovery rate of recombinant protein A and greatly reduced in cost. The method comprises the following steps: (1) heating a bacterial suspension containing recombinant protein A with a histidine tag so as to fully fragment bacteria; (2) adding polyethyleneimine and successively carrying out uniform mixing and centrifugation so as to obtain supernatant containing the recombinant protein A, wherein a weight ratio of the bacterial suspension containing recombinant protein A to polyethyleneimine is 10000: 1 to 100: 1; (3) allowing the supernatant to pass through a metal ion chelated chromatography column and flushing a medium by using a PBS buffer solution so as to obtain a pretreated recombination protein A solution; (4) removing a salt so as to obtain a desalted recombination protein A solution; and (5) purifying the desalted recombination protein A solution with an affinity chromatographic filling material so as to obtain the recombinant protein A solution without endotoxin, wherein the affinity chromatographic filling material can specifically bind to endotoxin.

Description

technical field [0001] The invention relates to a method for removing endotoxin in recombinant protein A solution, belonging to the technical field of bioengineering. Background technique [0002] Staphylococcal aureus protein A (Staphylococcal aureus Protein A, SPA), referred to as protein A, is a cell wall protein of Staphylococcus aureus. It was determined in 1959 that it can bind to immunoglobulin (Ig) substances, and then it was confirmed that it can reversibly specifically bind to the Fc fragment of IgG in human and various mammalian sera, and does not affect the binding activity of IgG to antigens , is widely used in scientific research, biological reagents, antibody purification in pharmaceutical companies and medical fields. [0003] Natural protein A only accounts for 1.7% of the total protein of the bacteria, the purification steps are cumbersome, the yield is low, and it is difficult to meet the needs of large-scale production, and Staphylococcus aureus is a pat...

Claims

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Application Information

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IPC IPC(8): C07K14/31C07K1/36C07K1/34C07K1/22
CPCC07K14/31
Inventor 卢晅宋炜
Owner 湖北中创医疗用品有限公司
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