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Method of separating and purifying recombinant stphylococcl protein A

A staphylococcal protein, separation and purification technology, applied in the field of separation and purification of recombinant staphylococcal protein A, can solve the problems of inconvenient operation, etc., and achieve the effect of reducing residual amount, reducing purification pressure, and combining tightly

Active Publication Date: 2017-07-25
GUANGZHOU KONCEN BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Patent CN103145813B mentions how to remove endotoxin, mainly using two activated carbon adsorption-hydrophobic chromatography, because the activated carbon is in powder form, the actual operation is not very convenient

Method used

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  • Method of separating and purifying recombinant stphylococcl protein A
  • Method of separating and purifying recombinant stphylococcl protein A
  • Method of separating and purifying recombinant stphylococcl protein A

Examples

Experimental program
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Effect test

Embodiment 1

[0040] 1) Cell pretreatment: take 1 kg of cells containing staphylococcal protein A (such as genetically engineered bacteria E. coli expressing staphylococcal protein A), add 10 L of Bis-tris buffer solution with a pH=7.2 and a concentration of 10 mM to suspend the bacteria The body was crushed twice with a high-pressure homogenizer at a pressure of 800 bar, and about 10 L of the supernatant was collected by centrifugation. The resulting solution was added with hydrochloric acid to adjust the pH to 2.0, centrifuged, and the supernatant was adjusted to pH = 6.5 with sodium hydroxide to obtain a protein solution.

[0041] 2) The first chromatographic column treatment: will be equipped with TOYOPEARL NH 2 The chromatographic column with -750F filler is equilibrated with 5 times the column volume with an equilibrium solution containing 10mM Bis-tris and 0.5wt% TritonX-100, 0.1mol / LNaCl, pH 6.5, and the protein solution obtained in step 1 is loaded onto the used TOYOPEARL NH equil...

Embodiment 2

[0049] 1) Bacteria pretreatment: take 1 kg of bacteria containing staphylococcal protein A (such as genetically engineered bacteria Escherichia coli expressing staphylococcal protein A), add 10 L of Bis-tris buffer solution with a pH=7.2 and a concentration of 30 mM to suspend the bacteria The body was crushed twice with a high-pressure homogenizer at a pressure of 800 bar, and about 10 L of the supernatant was collected by centrifugation. The resulting solution was added with hydrochloric acid to adjust the pH to 4.0, centrifuged, and the supernatant was adjusted to pH = 7.2 with sodium hydroxide to obtain a protein solution.

[0050] 2) The first chromatographic column treatment: will be equipped with TOYOPEARL NH 2 Equilibrate 5 times column volume with equilibrium solution (containing 20mM Bis-tris and 1wt% TritonX-100, 0.1mol / LNaCl, pH 7.0) for the chromatography column with -750F filler, and load the protein solution obtained in step 1 to the used equilibrium Liquid bal...

Embodiment 3

[0058] Basically the same as in Example 1, the difference is that step 1) cell pretreatment: take 1 kg of cell containing staphylococcal protein A, add 10 L of Bis-tris buffer solution with a pH=7.2 and a concentration of 20 mM to suspend the cell, Then use a high-pressure homogenizer to crush twice at a pressure of 800 bar, and collect about 10 L of supernatant by centrifugation. The resulting solution was added with hydrochloric acid to adjust the pH to 3.0, centrifuged, and the supernatant was adjusted to pH = 7.2 with sodium hydroxide, thereby obtaining a protein solution.

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Abstract

The invention discloses a method of separating and purifying recombinant stphylococcl protein A. A series of impurities such as endotoxin, residual proteins (HCP) of escherichia coli, residual DNA escherichia coli and protein aggregates are effectively removed by adopting stuffing of two mixing mechanisms by way of two-step chromatography and ultrafiltration. The purest target sample is obtained by fewest steps, so that the process control cost is lowered, and the method has the characteristics of being good in stability, clear in process control index, suitable for scaled industrial production the like. The protein purity of a purified protein solution is greater than 97% by means of high performance liquid and SDS-PAGE electrophoresis; the protein recovery rate is greater than 75%; the endotoxin is reduced to 1EU / mg below; HCP and residual DNA of escherichia coli are lower than 5ppm, and the recombinant stphylococcl protein A can be used as a raw material for synthesizing clinical staphylococcus protein A immunosorbent.

Description

[0001] Technical field: [0002] The invention relates to the technical field of biomedicine, in particular to a method for separating and purifying recombinant staphylococcal protein A. [0003] Background technique: [0004] Staphylococcal protein A (referred to as protein A or SPA) is a protein with a single-chain polypeptide structure, which contains five highly similar immunoglobulin Fc binding regions, and can specifically bind to human immunoglobulin (IgG). The SPA is coupled to the agarose carrier, and the carrier-SPA complex is prepared and loaded into a column to make a staphylococcal protein A immunoadsorption column. When human plasma passes through the column, the IgG pathogenic antibody in the plasma will be specifically Some diseases and symptoms caused by changes in the quality and quantity of IgG antibodies can be significantly treated and alleviated. Staphylococcal protein A immunoadsorption column has been widely used in the treatment of autoimmune diseases,...

Claims

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Application Information

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IPC IPC(8): C07K14/31C07K1/36C07K1/18C07K1/34
CPCC07K14/31
Inventor 张海珍余波光李乐军谢江明陈校园
Owner GUANGZHOU KONCEN BIOSCI
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