Unlock instant, AI-driven research and patent intelligence for your innovation.

A method for extracting and purifying recombinant human vascular endostatin

A technology of vascular endothelium and purification method, which is applied in the field of extraction and purification of recombinant human endostatin, can solve the problems of easy inactivation, low natural folding rate, and difficulty, etc., to promote dissolution, maintain and stabilize secondary structure, increase The effect of large chance of correct pairing

Active Publication Date: 2017-04-26
DENOVO BIOPHARMA HANGZHOU LTD
View PDF2 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the low natural folding rate of human endostatin, most of the disulfide bonds need to be formed in the process of refolding, and human endostatin has two pairs of disulfide bonds, and both pairs of disulfide bonds need to be refolded. Correct pairing can refold into biologically active endostatin. In addition, the secondary structure of the protein itself is variable, and it is not easy to form the correct disulfide bond. Misfolding and polymerization often result in low refolding yield, so , it is difficult to achieve a high refolding yield
At the same time, endostatin itself is slightly acidic, its activity and solubility are unstable and easy to inactivate in neutral and alkaline environments, and its sensitivity to the environment also increases the realization of recombinant human endostatin to a certain extent. The difficulty of high renaturation yield makes the yield of recombinant human endostatin target product lower

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A method for extracting and purifying recombinant human vascular endostatin
  • A method for extracting and purifying recombinant human vascular endostatin
  • A method for extracting and purifying recombinant human vascular endostatin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The extraction and purification method of the recombinant human vascular endostatin of the present implementation comprises the following steps:

[0039] (1) engineering bacterium fermentation: after getting engineering bacterium to amplify in LB culture medium, press the 6% inoculum amount of fermentation volume, inoculate to the fermentation medium in the fermentor tank that specification is 50L, the fermented liquid volume is 44L, ferment to OD 600 The value is 12.5. Then, add 3.75g of inducer IPTG, 13g / L of carbon source substance glucose, and 5g / L of nitrogen source substance yeast extract to it, cultivate at 30°C for 2.5h, and then add 2.4g The inducer IPTG continued to culture for 4h.

[0040] Wherein, the fermentation medium is composed of 10g / L tryptone, 20g / L yeast extract, 12.5g / L hydrolase protein, 1g / LKH 2 PO 4 , 10g / L glucose composition.

[0041] (2) engineering bacterium is broken: the bacterium liquid that obtains in step 1) is washed with the 20mmo...

Embodiment 2

[0055] The extraction and purification method of the recombinant human vascular endostatin of the present implementation comprises the following steps:

[0056] (1) engineering bacterium fermentation: first get engineering bacterium after amplifying in LB substratum, press the 5% inoculum size of fermentation volume, inoculate to the fermentation medium in the fermentor tank that specification is 50L, the fermented liquid volume is 44L, Ferment to OD 600 The value is 10, then, add 2.49g of inducer IPTG, 15g / L of carbon source glucose, and 6g / L of nitrogen source yeast extract, culture at 34°C for 3h, and then add 3.75g of inducer IPTG, continue to culture for 3h.

[0057] Wherein, the fermentation medium is composed of 5g / L tryptone, 25g / L yeast extract, 7.5g / L hydrolase protein, 9g / LKH 2 PO 4 , 20g / L glucose composition.

[0058] (2) engineering bacterium is broken: the bacterium liquid that obtains in step 1) is washed with the 20mmol / L tris-hydrochloric acid (Tris-HCl) ...

Embodiment 3

[0072] The extraction and purification method of the recombinant human vascular endostatin of the present implementation comprises the following steps:

[0073] (1) engineering bacterium fermentation: get engineering bacterium after amplifying in LB medium, inoculate into the fermentation medium in the fermentor tank that specification is 50L by 8% inoculum of fermentation volume, fermented liquid volume is 44L, ferment to OD 600 The value is 15, then, add 4.99g of inducer IPTG, 12g / L carbon source substance glucose, and 4g / L nitrogen source substance yeast extract to it, cultivate at 37°C for 3.5h, and then add 1.25g of inducer Agent IPTG, continue to culture for 5h.

[0074] Wherein, the fermentation medium is composed of 15g / L tryptone, 15g / L yeast extract, 17.5g / L hydrolase protein, 5g / LKH 2 PO 4 , 15g / L glucose composition.

[0075] (2) engineering bacterium is broken: the bacterium liquid that obtains in step 1) is washed with the 20mmol / L tris-hydrochloric acid (Tri...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
concentrationaaaaaaaaaa
concentrationaaaaaaaaaa
crush indicatorsaaaaaaaaaa
Login to View More

Abstract

The invention relates to a method for extracting and purifying recombinant human endostatin. The method comprises the following steps of fermenting engineering strains, crushing the engineering strains, washing inclusion bodies, carrying out denaturing purification on the recombinant human endostatin, renaturing the denatured recombinant human endostatin, standing the renatured recombinant human endostatin and carrying out chromatographic separation on the recombinant human endostatin. According to the method, the condition that efficient induced expression is achieved at relatively high thallus concentration can be realized during the fermentation of the engineering strains, and a relatively high renaturation ratio is achieved during the renaturation of the denatured recombinant human endostatin, so that the large-scale production of the recombinant human endostatin is realized.

Description

technical field [0001] The invention belongs to the field of extraction and purification of recombinant proteins, and in particular relates to an extraction and purification method of recombinant human endostatin. Background technique [0002] Human vascular endostatin is currently the most effective inhibitor of tumor angiogenesis with the best experimental effect. Experiments have shown that endostatin can inhibit vascular endothelial cells and growing blood vessels, and can better inhibit tumor growth and metastasis. In terms of its mechanism of action, human endostatin inhibits the blood supply of tumor tissue in the human body, causing the tumor to lack nutrients and oxygen and stop growing, and then gradually shrink until death. , has the advantage of no obvious toxic and side effects, and thus has received extensive attention from the medical community. [0003] At present, human endostatin is mainly prepared by obtaining engineering bacteria carrying human endostat...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/02C07K1/18C07K1/16C07K1/14
Inventor 荣志刚姚建林赵唯一黄佩华朱浩文陈卫崔志远徐霞
Owner DENOVO BIOPHARMA HANGZHOU LTD