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Method for separating and purifying annexin V or derivative thereof expressed by escherichia coli

A technology for annexin and Escherichia coli, applied in the field of separation and purification of annexin V or its derivatives, can solve the problems of low crushing efficiency, high production cost, limited use times, etc., achieve good crushing effect and reduce production cost , The effect of reducing the cost of crushing

Active Publication Date: 2015-04-22
CHANGZHOU QIANHONG BIOPHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the heparin affinity chromatography column used in the first method is expensive and has a limited number of uses, resulting in high production costs and is not suitable for industrial production
The purification effect of the second and third methods is not good, and the purity of the obtained annexin V is difficult to meet the quality requirements of biological products
[0007] The enzymatic fragmentation method has the best effect among the non-mechanical fragmentation methods, but the cost is still high due to the need for enzyme reagents, and the subsequent purification needs to remove the added enzyme reagents, which increases the pressure of subsequent purification
[0008] The advantage of the osmotic pressure impact crushing method is that the conditions are milder and the cost is low; the disadvantage is that the crushing efficiency is low, and it is generally used for strains that do not have cell walls or have fragile cell walls
The advantage of repeated freeze-thaw crushing method is that the cost is low, and the disadvantage is that the crushing efficiency is low, and it is generally used for bacteria with fragile cell walls.
[0009] The cell wall of Escherichia coli is composed of strong peptidoglycan. Therefore, the traditional method of crushing E. coli cells mainly adopts the ultrasonic crushing method in the mechanical crushing method or the enzymatic crushing method in the non-mechanical crushing method. Although their crushing effect is relatively Good, but more expensive
In particular, the ultrasonic disruption method is easy to release a large amount of impurity proteins while breaking the bacteria and releasing the target protein, which increases the pressure of subsequent purification.
[0010] At present, there is no literature report on the combination of two physical crushing methods to crush E. coli cells

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1）

[0020] This embodiment is a method for separating and purifying Annexin V expressed in Escherichia coli, which has the following steps:

[0021] ①Bacteria-breaking process: 100 g of E. coli cells expressing Annexin V collected by fermentation and centrifugation were first frozen at -20°C, then thawed at 20°C, and then the aforementioned freezing and thawing was repeated twice. The Escherichia coli cells after repeated freezing and thawing were thoroughly mixed with sucrose hypertonic solution (composed of 65g sucrose, 10mmol / L disodium ethylenediaminetetraacetic acid and 100mL water), and statically placed at 25℃. Then it was diluted with water to 2L, and stirred for 1 hour until it was fully broken, and the supernatant (that is, the broken bacteria liquid) was collected by centrifugation, and the broken wall was checked by an optical microscope. The amount of bacteria in front of the wall × 100%).

[0022] ②Coarse extraction process: Add ammonium sulfate to the bacteriostasi...

Embodiment 2）

[0027] Step 1. and step 2. of this embodiment are the same as in Embodiment 1, the difference being:

[0028]③ Anion exchange chromatography: Load the crude extract obtained from the crude extraction process onto an anion exchange chromatography column that has been equilibrated with buffer B, then wash with buffer B, and then elute with buffer C , collect the anion exchange eluent; the buffer B is an ammonium chloride solution with a pH value of 6.0, wherein the concentration of ammonium chloride is 60mmol / L; the buffer C is an ammonium chloride / Sodium chloride mixed solution, wherein the concentration of ammonium chloride is 60mmol / L, and the concentration of sodium chloride is 0.1mol / L. The filler in the anion exchange chromatography column is Q Sepharose FF.

[0029] 4. Hydrophobic chromatography: Add ammonium sulfate to the anion exchange eluent obtained in step 3. until the concentration of ammonium sulfate is 1mol / L, and adjust the pH value of the solution to 7.0 to o...

Embodiment 3）

[0032] This example is a method for the separation and purification of Annexin V derivatives expressed in Escherichia coli. The Annexin V derivatives are connected with Gly-Pro-Arg-Pro at the N-terminal of Annexin V by genetic engineering methods, and at the same time A protein obtained by linking the C-terminal of annexin V to the C-terminal domain of hirudin. The separation and purification method has the following steps:

[0033] ① Bacteria-breaking process: 100 g of E. coli cells expressing the above-mentioned annexin V derivatives collected by fermentation and centrifugation were first frozen at -25°C, then thawed at 25°C, and then repeated freezing and thawing for 3 Second-rate. The Escherichia coli cells after repeated freezing and thawing were thoroughly mixed with sucrose hypertonic solution (composed of 65g sucrose, 10mmol / L disodium ethylenediaminetetraacetic acid and 100mL water), and statically placed at 20℃. Then it was diluted with water to 2L, and stirred for...

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Abstract

The invention discloses a method for separating and purifying annexin V or a derivative thereof which is expressed by escherichia coli. According to the method, a cell lysis technology, a rough extracting technology and a refining technology are sequentially carried out on the escherichia coli by which the annexin V or the derivative thereof is expressed. The cell lysis technology comprises the following steps: first repeatedly carrying out freeze lysis breaking and then carrying out osmotic pressure impact breaking. The refining technology comprises the following steps: first carrying out hydrophobic chromatography and then carrying out anion exchange chromatography, or first carrying out anion exchange chromatography and then carrying out hydrophobic chromatography, wherein agarose which is associated with a DEAE (diethyl-aminoethanol) ligand or a Q ligand is used as a filler during the anion exchange chromatography, or polymethacrylate which is associated with the DEAE ligand or the Q ligand is used as the filler during the anion exchange chromatography; the agarose which is associated with phenyl or butyl or octyl is used as the filler during the hydrophobic chromatography, or the polymethacrylate which is associated with the phenyl or the butyl or the octyl is used as the filler during the hydrophobic chromatography. The separating and purifying method is low in cost, and the purity of the obtained target protein is greater than or equal to 95%.

Description

technical field [0001] The invention relates to a method for separation and purification, in particular to a method for separation and purification of annexin V expressed in Escherichia coli or derivatives thereof. Background technique [0002] The Annexins family is a class of calcium-dependent phospholipid-binding proteins. Annexin V is one of the most widely distributed and abundant members of the annexin family. Annexin V has a variety of important physiological functions, such as anticoagulant activity, calcium ion channel activity, inhibition of phospholipase A2 and phospholipase C activity, and so on. At the same time, annexin V plays an important role in the process of cell secretion regulation and signal transduction, and it also has a very high affinity for activated platelets and apoptotic cells. [0003] There are three methods for the purification of annexin V expressed in early Escherichia coli: (1) Heparin affinity chromatography. (2) Carry out cation excha...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/47C07K1/36C07K1/30C07K1/20C07K1/18
CPCC07K14/47
Inventor 张笋华潘伟忠于洋刘军韦利军
Owner CHANGZHOU QIANHONG BIOPHARMA