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Method for separating isomerism protein from recombinant human ciliary neurotrophy factor and its mutant

A ciliary neurotrophic and mutant technology, which is applied in the direction of animal/human protein, growth factor/inducible factor, specific peptide, etc., can solve the problems of affecting drug efficacy, producing antibodies, and heterogeneous protein has no curative effect, so as to improve the therapeutic effect. Effect, the effect of reducing antibody titer

Inactive Publication Date: 2008-01-30
兰州生物制品研究所
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Problems solved by technology

The recombinantly expressed human ciliary neurotrophic factor and its mutants can be highly expressed in Escherichia coli, but the heterogeneous protein present in it has no curative effect, instead it will produce antibodies, affecting its actual drug efficacy

Method used

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  • Method for separating isomerism protein from recombinant human ciliary neurotrophy factor and its mutant
  • Method for separating isomerism protein from recombinant human ciliary neurotrophy factor and its mutant
  • Method for separating isomerism protein from recombinant human ciliary neurotrophy factor and its mutant

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Embodiment Construction

[0010] Fermentation culture and induced expression of recombinant human ciliary neurotrophic factor (CNTF)

[0011] The recombinant CNTF full gene synthesis of the present invention is completed by Shanghai Shenggong Biological Engineering Technology Service Co., Ltd. Take the synthesized CNTF and culture it by fermentation. The compound medium used is composed of: peptone 20g / L, yeast powder 10g / L, and sodium chloride 5g / L.

[0012] The fermentation seed liquid (ie CNTF) is connected to the fermentation medium at a ratio of 1:10~1:40 (V / V), and the appropriate fermentation culture temperature, pH value, and dissolved oxygen DO are controlled. One hour sampling to measure OD 600 Value when OD 600 At 5-6 hours, add isopropyl-β-D-thiogalactoside (IPTG) to induce expression. The final concentration of IPTG is 0.1mmol / L. After 4 hours of induction, the culture is terminated, and the culture solution is collected by centrifugation at 4000g Bacteria.

[0013] Refolding of CNTF protein i...

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Abstract

The invention relates to a method to separate isomeric protein form gene recombinant expression protein. The method of the invention is that: the collected wet bacterium is made into bacterium soliquoid in a cleaning buffer liquid, and shattered and decentered by ultrasonic, and the clear liquid is abandoned, the precipitation is cleaned by TE buffer liquid containing urea and TritonX-100, then protein endosome is acquired, and the endosomes is fully dissolved by a buffer liquid containing guanidine hydrochloride or urea; the dissolving liquid is diluted and renatured by the buffer liquid containing Tris-HCl, arginine and EDTA, then stand for 24-48h, and becomes a CNTF renatured protein liquid; after being ultrafiltered and concentrated, the renatured protein is transferred into the Tris buffer liquid through gel filtration chromatography to collect protein peak; finally the CNTF renatured protein liquid with transferred liquid is sent to Q sephrose-Fast Flow anion exchange column, to remove unrenatured CNTF proteins and other proteins.

Description

Technical field [0001] The present invention relates to a method for separating heterogeneous proteins from proteins expressed by genetic recombination, more specifically, a method for separating heterogeneous proteins from recombinant human ciliary neurotrophic factor or its mutants. Background technique [0002] In the prokaryotic expression protein of genetic recombination, the primary structure is completely correct, but there is no biologically active isomer due to the problem of protein renaturation. Due to the existence of isomers, its biological activity is greatly affected. Therefore, the separation of isoforms in recombinantly expressed proteins is a problem that needs to be solved in biopharmaceuticals. Human ciliary neurotrophic factor (CNTF) can be used to treat common obesity and diabetic obesity. The recombinantly expressed human ciliary neurotrophic factor and its mutants can be highly expressed in E. coli, but the heterogeneous proteins present in them have no cu...

Claims

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Application Information

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IPC IPC(8): C07K14/475C07K1/36C07K1/14
Inventor 蒋琳应莲芳谢溱高雪峰梁凌宇李萍卜晓萍马亚茹
Owner 兰州生物制品研究所
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