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Extraction and purification process for recombinant protein

A technology of recombinant protein and purification method, applied in the field of biopharmaceuticals

Active Publication Date: 2012-09-05
SUZHOU SIXTH PHARMA PLANT OF JIANGSU WUZHONG PHARMA GROUP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The object of the present invention is to provide a recombinant protein extraction and purification method, which can effectively solve the low purity, low efficiency and production cost of the final product of the existing recombinant protein extraction and purification method advanced defects

Method used

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  • Extraction and purification process for recombinant protein
  • Extraction and purification process for recombinant protein
  • Extraction and purification process for recombinant protein

Examples

Experimental program
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Effect test

no. 1 example

[0079] Step 101, get the engineering bacteria, carry out amplification with bacteriolysis broth medium (LB medium), put the engineering bacteria liquid of 10% of the fermentation volume into a fermenter containing high-density formula medium for fermentation, high-density formula The medium is 10g / L tryptone, 20g / L yeast extract, 12.5g / L hydrolase protein, 4g / L KH 2 PO 4 , 15g / L glucose;

[0080] Step 102, during the fermentation process, regularly and continuously drop increasing amounts of high-density formula culture medium;

[0081] Step 103, after 7.5 hours of fermentation, induce the engineered bacteria at a temperature of 42° C. for 3.5 hours, and release the fermented bacterial liquid from the fermenter after 11 hours of fermentation;

[0082] Step 104, take 100 g of engineering bacteria wet bacteria, add 1000 ml of the first buffer solution (20 mmol / LTris-HCl (tris-hydrochloric acid), pH 8.0), wash the bacteria, and centrifuge at 4 ° C for 10 minutes, Rotate at 700...

no. 2 example

[0099] Step 201, get engineering bacterium, after carrying out amplification with bacteriolysis broth culture medium (LB medium), insert the engineering bacterium of 10% by fermentation volume in the fermenter that contains high-density formula medium to carry out fermentation; High-density formula The medium is 5g / L tryptone, 15g / L yeast extract, 7.5g / L hydrolase protein, 1g / L KH 2 PO 4 , 10g / L glucose;

[0100] Step 202, during the fermentation process, regularly and continuously drop increasing amounts of high-density formula culture medium;

[0101] Step 203, after 8 hours of fermentation, inducing the engineered bacteria at a temperature of 42°C for 3 hours, and releasing the fermented bacterial liquid from the fermenter after 11 hours of fermentation;

[0102] Step 204, take 100 g of engineering bacteria wet bacteria, add 500 ml of the first buffer solution (20 mmol / LTris-HCl (tris-hydrochloric acid), pH 8.0), wash the bacteria, and centrifuge at 4 ° C for 10 minutes, ...

no. 3 example

[0119] Step 301, get the engineering bacteria, after amplifying with the bacteriolysis broth medium (LB medium), insert the engineering bacteria liquid according to 10% of the fermented body into the fermenter containing the high-density formula medium to enter the fermentation, the high-density Formula medium is 15g / L tryptone, 25g / L yeast extract, 17.5g / L hydrolase protein, 9g / L KH 2 PO 4 , 20g / L glucose;

[0120] Step 302, during the fermentation process, regularly and continuously drop increasing amounts of high-density formula culture medium;

[0121]Step 303, after 7 hours of fermentation, inducing the engineered bacteria at a temperature of 42°C for 4 hours, and releasing the fermented bacterial liquid from the fermenter after 11 hours of fermentation;

[0122] Step 304, take 100 g of engineering bacteria wet bacteria, add 800 ml of the first buffer solution (20 mmol / LTris-HCl (tris-hydrochloric acid), pH 8.0), wash the bacteria, and centrifuge at 4 ° C for 10 minutes...

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Abstract

The invention relates to a recombinant protein extraction and purification method, comprising engineering bacteria fermentation, engineering bacteria fragmentation, inclusion body washing, recombinant protein denaturation and purification, second recombinant protein denaturation and purification, denaturized recombinant protein renaturation, renaturated recombinant protein placing, ion exchange chromatography and molecular sieve chromatography, wherein, the renaturation recombinant protein is placed for 5 to 8 days under the temperature of 2 to 8 DEG C. The recombinant protein extraction and purification method improves the total yield of the recombinant protein, correspondingly improves the purity and efficiency of the final products, reduces the production cost and is beneficial to large scale industrial production.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to a method for extracting and purifying recombinant proteins expressed by genetic engineering bacteria. Background technique [0002] The recombinant proteins expressed by many genetically engineered bacteria (Escherichia coli) mostly (all) exist in the form of inclusion bodies, such as: interleukin-2, human granulocyte stimulating factor, human granulocyte macrophage stimulating factor fusion protein, etc. Since the quality and efficiency of the method of extracting and purifying recombinant protein from inclusion bodies directly affect the quality and efficiency of the final product, and then affect the economic benefits, this method of extraction and purification has always been highly valued. [0003] In the recombinant protein extraction and purification method, the fermentation conditions of engineered bacteria are one of the key technologies that affect the amount of bacter...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/14C07K1/18C07K1/16
Inventor 荣志刚姚建林钟慎政赵唯一朱浩文汪志超顾培诚
Owner SUZHOU SIXTH PHARMA PLANT OF JIANGSU WUZHONG PHARMA GROUP
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