Method for renaturing and purifying novel thrombopoietin

A technique for thrombopoietin and purification method, which is applied in the field of renaturation and purification of novel thrombopoietin mimetic peptides, and achieves the effects of easy industrialization and amplification, simple operation and reasonable process combination.

Active Publication Date: 2012-01-18
山东泉港药业有限公司
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AI Technical Summary

Problems solved by technology

However, there is currently no production process suitable for large-scale production of thrombopoietin peptidomimetics

Method used

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  • Method for renaturing and purifying novel thrombopoietin
  • Method for renaturing and purifying novel thrombopoietin
  • Method for renaturing and purifying novel thrombopoietin

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Embodiment Construction

[0028] The invention provides a novel thrombopoietin peptidomimetic refolding and purification method. After high-density fermentation of Escherichia coli engineering bacteria containing the thrombopoietin peptidomimetic gene, the bacterial body is washed after taking it out: melting, adding pre-cooled TE buffer, stir well, centrifuge at 4000 rpm for 20 minutes at a temperature of 4°C, discard the supernatant to save the precipitate, and remove the residual medium components in the fermentation medium; then proceed to the following steps:

[0029] (1) Ultrasonic bacteria destruction: add 10ml pre-cooled TE buffer solution per gram of bacteria, the buffer is 50mM Tris-Hcl and 5mM EDTA, the pH of the buffer is 8.5, after mixing, add lysozyme until the concentration of the bacteria is 1%, mix well, keep the temperature at 2-8°C, and act for more than 1 hour; ultrasonically break bacteria for 2min / time, 6 times in total; then centrifuge at 8500 rpm for 15 minutes at 2-8°C, Collect...

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Abstract

The invention discloses a method for renaturing and purifying novel thrombopoietin mimetic peptide, the method comprises the following steps: performing a high density fermentation to escherichia coli engineering bacteria containing thrombopoietin mimetic peptide, carrying out an ultrasonication to thalline, urea cracking, renaturing, regulating the pH value, combining and purifying through Protein-AMabselectSuRe chromatography, SPSepharoseH.P chromatography and Sephacry1S-200 molecular sieve chromatography, and preparing thrombopoietin mimetic peptide protein which conforms to the Chinese pharmacopoeia specification. The beneficial effects of the present invention have high renaturation rate, reasonable purifying process combination, simplified operation program, high yield and low production cost, and the method provides the possibility for the large scale production of the thrombopoietin mimetic peptide.

Description

[0001] technical field [0002] The invention relates to a production method of a thrombopoietin mimic, in particular to a method for refolding and purifying a novel thrombopoietin mimic expressed in Escherichia coli. [0003] [0004] Background technique [0005] Chronic idiopathic thrombocytopenic purpura (ITP) is an autoimmune disease caused by an autoimmune reaction that causes thrombocytopenia, characterized by low platelet count, and its patients are at high risk of bleeding, often resulting in small Vascular bleeding with symptoms of bruising, nosebleeds, bleeding gums, and in severe cases fatal gastrointestinal and intracerebral bleeding. At present, the main means of treatment of ITP is to use immunosuppressive drugs, such as corticosteroids, intravenous immunoglobulin, anti-D immunoglobulin and Genentech's product rituximab, and the last resort for severely debilitating patients is Splenectomy. Most of these therapeutic drugs have not been investigated in rando...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/475C07K1/16C07K1/14
Inventor 宋磊陈文芳刘宁刘红军
Owner 山东泉港药业有限公司
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