Large-scale cytorrhyctes compound purifying technology for recombining tPA derivative

A technology of inclusion body and volume, applied in the field of bioengineering, can solve the problems of lack and insufficient production technology

Inactive Publication Date: 2005-05-11
上海中科伍佰豪生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, most genetically engineered drugs have been successfully imitated in my country. As tPA with great commercial value, it has not been successfully imitated so far. A very important factor is the lack of production technology research and lack of

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Refolding of inclusion bodies of tissue plasminogen activator

[0063] The purified tPA inclusion bodies with a wet weight of 10 g were dissolved in 1000 ml of denaturing solution (8M urea or 7M guanidine hydrochloride, 20 mM DDT), stirred at room temperature for 2-5 hours, and then centrifuged at 15,000 rpm for 50 minutes.

[0064] Dilute the centrifuged supernatant into 8-12 liters of refolding solution I (3-7M urea, 0.5-1M arginine, 50mM Tris HCl, pH8.2-9.0, 1-10mM reduced glutathione GSH, 0.05-5mM oxidized glutathione (GSSG). After stirring overnight at room temperature, filter through a 0.45 μm filter.

[0065] Dilute the filtrate into 80-120 liters of refolding solution II (0.1-2.5M urea, 0.3-1M arginine, 50mM Tris·HCl, pH8.2-9.0). Refold at room temperature for 16-20 hours.

[0066] Filtrate (membrane specification 0.22um), and concentrate the filtrate to 3-10 liters by ultrafiltration with a 10K-Millipore ultrafiltration membrane to obtain the first concentra...

Embodiment 2

[0070] Purification of inclusion bodies of tissue plasminogen activator

[0071] In this example, the second concentrated solution containing tissue plasminogen activator prepared in Example 1 was purified by conventional ion column chromatography.

[0072] method:

[0073] Put the renatured sample obtained in Example 1 on the Pharmacia SP-Sepharose Fast Flow column, and then use the eluting solution I (50mM ammonium acetate pH4.0-5.0 containing 0-1M NaCl) to elute after loading, and then elute with Solution II (containing 0-1M arginine, 0.1-1M NaCl in 50mM ammonium acetate pH4.5-6.0) and eluting solution III (containing 0.1-1M arginine, 0.1-1M NaCl in 50mM ammonium acetate pH5.5 -7.0) eluted to obtain purified recombinant tPA derivatives.

[0074] result:

[0075] Finally, about 150-200 mg of purified recombinant tPA derivatives with a specific activity of 40,000-200,000 IU / mg protein were obtained. The amount of glutathione used in the refolding process has been reduced ...

Embodiment 3-5

[0077] Refolding of inclusion bodies of tissue plasminogen activator

[0078] Repeat Example 1, the difference is to change part of the renaturation conditions in Example 1, as shown in Table 1.

[0079] Example 1

Example 3

Example 4

Example 5

First

Refolding

liquid

Glutathione concentration

6mM

1.5mM

12mM

15mM

Reduced Glutathione:

oxidized glutathione

5∶1

6∶1

5∶1

4∶1

denaturant concentration

3M urea

5M urea

5M Guanidine Hydrochloride

6M Guanidine Hydrochloride

Arginine concentration

0.5M

0.5M

0.5M

1M

pH

8.2-8.5

8.0

8.5

7.8

second

Refolding

liquid

pH

8.2-8.5

8.5

8.0

8.8

denaturant concentration

0.2M urea

0.5M urea

1M urea

2.5M urea

Arginine concentration

0.3M

0.3M

0.5M

1M

[0080] r...

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Abstract

This invention relates to protein renaturation. It is used for large inclusion body for recombination of tPA derivative and is carried out by: dissolving inclusion body of target protein, centrifuging to obtain supernatant containing it, diluting it with first renaturation liquid containing oxidation reducing pairs, settling a period, diluting it with second renaturation liquid without oxidation reducing pairs, settling a period, filtrating and concentrating to obtain primary concentrated liquid, filtrating and diluting with buffering liquid to regulate pH value 4.0-5.5, filtrating and concentrating to obtain secondary liquid containing renatured target protein.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to the technique of refolding and purifying the inclusion body of tissue plasminogen activator. Background technique [0002] The inclusion body denaturation and renaturation of tissue plasminogen activator expressed in Escherichia coli is a difficult problem in the renaturation and purification of protein inclusion bodies. Due to the existence of a large number of disulfide bonds in protein molecules, the renaturation and purification process is relatively complicated and the cost is high. The cost of the purification process directly leads to the high price of drugs. [0003] For the recombinant protein expressed in Escherichia coli in the form of inactive inclusion bodies, the commonly used refolding method is to denature and dissolve it with high concentration of guanidine hydrochloride or urea, and then obtain the refolded recombinant protein by removing the denaturant. [0004]...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/113C12N9/00C12N9/72
Inventor 张毅陆坚峰杨胜利褚仲梅刘成屈贤铭
Owner 上海中科伍佰豪生物工程有限公司
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