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Preparation of recombinant human nerve growth factor ( rhNGF ) protein and renaturation solution

A technology of nerve growth factor and protein, which is applied to a method for preparing recombinant human nerve growth factor protein and the field of refolding solution, which can solve the problems of complicated preparation process, high production cost, and low body refolding rate, and achieve operational Simple, short time-consuming, simple equipment requirements

Active Publication Date: 2012-08-08
ZHEJIANG NUPTEC BIOMACROMOLECULAR PURIFICATION RESINS MFG INC LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Aiming at the problems of low inclusion body renaturation rate, complex preparation process, long time-consuming, complicated operation and high production cost of rhNGF protein in the engineering E. , short time-consuming, easy-to-operate renaturation method

Method used

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  • Preparation of recombinant human nerve growth factor ( rhNGF ) protein and renaturation solution
  • Preparation of recombinant human nerve growth factor ( rhNGF ) protein and renaturation solution
  • Preparation of recombinant human nerve growth factor ( rhNGF ) protein and renaturation solution

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Embodiment 1

[0177] 1. The activated BL21(DE3) / pET-28a-hNGF expression strain was cultured at 37°C for 4 hours with 1% inoculum size of 500ml LB (containing 50μg / ml kanamycin), and added IPTG (final concentration of 1mM ) was induced at 37°C for 5 hours.

[0178] 2. Centrifuge at 8000rpm for 5min to collect BL21(DE3) cells induced to express rhNGF protein, suspend the collected cells with 100ml of 50mM Tris-HCl (pH 8.0) and then sonicate: ultrasonication for 3s, interval of 5s, ultrasonication 100 times, The ultrasonic power is 650×0.45W.

[0179] 3. The sonicated cells were centrifuged at 12,000 rpm for 10 minutes to collect rhNGF protein inclusion bodies.

[0180] 4. The rhNGF protein inclusion body was washed once with 100ml washing buffer (50mM Tris-HCl, 2mM EDTA, 0.1M NaCl, 2% Triton X-100, 1mM DTT, pH8.0), centrifuged at 12000rpm for 10min, and 100ml inclusion body was used for precipitation Denaturing solution (50mM Tris-HCl, 8M urea, 2mM EDTA, 2mM DTT, pH 8.0) was dissolved for 2...

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Abstract

The present invention relates to the preparation of a recombinant human nerve growth factor (rhNGF) protein and a renaturation solution. The method includes steps of: expressing rhNGF protein by engineering Escherichia coli; breaking the engineering Escherichia coli bacteria expressing the rhNGF protein; centrifuging and collecting rhNGF protein inclusion body; carrying out denaturation dissolving on the inclusion body by using a denaturation system to form an inclusion body dissolved solution; adding a renaturation solution with a flow greater than 1080ml / min into the inclusion body dissolved solution for renaturation; and purifying the rhNGF protein. The renaturation solution contains 45-55mM Tris-HCl, 0-10mM beta- mercaptoethanol, 0-1mM cupric salt and 0.95-1.05mM EDTA, and has a pH of 8.5-10. The method of the invention utilizes instantaneous addition of renaturation solution for dilution and renaturation to enhance renaturation rate and reduce the production cost, and is simplefor operation.

Description

technical field [0001] The invention belongs to recombinant protein medicine in the field of medicine, and relates to a method for preparing recombinant human nerve growth factor protein and a refolding solution. Background technique [0002] Nerve growth factor (NGF) is one of the most important bioactive molecules in the nervous system. NGF has a nutritional effect on normal nerve cells, has a regulating effect on the repair function of damaged nerves, can maintain the survival of sympathetic nerves and sensory nerves, promote the differentiation of nerve cells, determine the extension direction of axons, and promote the development of the brain and the nervous system. It plays a decisive role in the growth, regeneration and functional recovery of damaged nerves. [0003] Human nerve growth factor (hNGF) can be applied to nervous system diseases, such as diabetic peripheral neuropathy, Alzheimer's disease, Parkinson's disease, facial neuritis, and including spinal cord in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C07K14/48C07K1/14
Inventor 钱永常
Owner ZHEJIANG NUPTEC BIOMACROMOLECULAR PURIFICATION RESINS MFG INC LTD
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