Method for preparing protein inclusion body and recombinant human beta-neural growth factor

A technology for nerve growth factor and inclusion body, which is applied in the field of protein inclusion body and the preparation of recombinant human β-nerve growth factor, can solve the problems of low dilution and renaturation rate, unfavorable industrialized production, large renaturation volume, etc. , to achieve the effect of improving renaturation rate, reducing process cost and realizing automation

Pending Publication Date: 2019-08-06
重庆科润生物医药研发有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The renaturation rate of dilute renaturation is low, the renaturation time is long, and a large renaturation volume is required when it is used in production, which is not conducive to industrial production, and the production cost is high

Method used

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  • Method for preparing protein inclusion body and recombinant human beta-neural growth factor
  • Method for preparing protein inclusion body and recombinant human beta-neural growth factor
  • Method for preparing protein inclusion body and recombinant human beta-neural growth factor

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preparation example Construction

[0042] The preparation method of recombinant human β-nerve growth factor disclosed in some embodiments of the present invention comprises the following steps:

[0043] Step (1) performing chromatographic renaturation on the inclusion body of the recombinant human β-nerve growth factor precursor protein containing an enterokinase cleavage site to obtain the renatured precursor protein;

[0044] Step (2) performing enterokinase digestion on the renatured precursor protein to obtain an enzymatic hydrolysis product;

[0045] Step (3) separating and purifying the enzymatic hydrolysis product.

[0046]In the above examples, the chromatographic renaturation technology is applied to the renaturation of rhpro-NGF inclusion bodies. Compared with the traditional dilution renaturation method, it realizes renaturation under high concentration conditions, reduces the operation volume, and makes the renaturation process and purification process Completed under the same operating unit, the r...

Embodiment 1

[0074] Example 1: Construction of engineering bacteria expressing rhpro-NGF containing an enterokinase cleavage site

[0075] Referring to the codon preference of Escherichia coli, without changing the amino acid sequence, optimize the rhpro-NGF base sequence, and insert the base of the enterokinase recognition site DDDDK between the leader peptide and the mature peptide base sequence of the rhpro-NGF protein base sequence. Add the NdeI restriction site to the 5' end of the optimized base sequence, add the NotI restriction site to the 3' end, and add the stop codon sequence TAATAA between the 3' end of the target protein and the NotI restriction site, The base sequence shown in SEQ ID NO:1 was obtained, and the encoded amino acid sequence was shown in SEQ ID NO:2. The designed sequence is synthesized by chemical synthesis. Digest the synthetic sequence with NdeI / NotI, connect it with the expression plasmid pET-30a(+) cut with the same enzyme, transform Escherichia coli DH5α ...

Embodiment 2

[0080] Example 2: High-density culture of rhpro-NGF engineering bacteria and induced expression of target protein

[0081] Streak the rhpro-NGF engineered bacteria on LB plates (kan 100mg / L) and culture them in a constant temperature incubator at 37°C for about 16-18 hours until a single colony grows. Pick a single colony of engineered bacteria and inoculate in 20ml LB medium (kan 100mg / L), and culture at 37□, 230rpm for 8h. Transfer 0.1% to 250ml LB (kan 100mg / L), 1L Erlenmeyer flask, culture at 37°C, 230rpm for 13h. Four bottles were cultivated in parallel, 1000ml of bacterial solution was prepared, and 5% was inoculated into the medium of the upper tank of the fermenter NLF-2220L. Before inoculation, the pH was adjusted to 7.0 with ammonia water, and the temperature during the fermentation process was controlled at 36□. The pH value and dissolved oxygen of the culture medium are controlled by adding ammonia water and increasing the stirring speed and ventilation, and the d...

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Abstract

The invention relates to a method for preparing a protein inclusion body and a recombinant human beta-neural growth factor. According to the invention, the inclusion body of the recombinant human beta-neral growth factor precursor protein containing the enterokinase enzyme sites is subjected to chromatographic renaturation to obtain a renaturation precursor protein, which is the protein inclusionbody; the renaturation precursor protein is digested with enterokinase to obtain an enzymatic hydrolysate; and the enzymatically hydrolyzed product is isolated and purified. The method applies a chromatographic method to the renaturation of the rhpro-NGF inclusion body, and significantly improves the renaturation rate and shortens the renaturation time compared with the traditional dilution renaturation method, so that the subsequent purification is simpler, and a high-purity product having a correct structure is obtained, the method can reduce the chance of protein aggregation, breaks the balance of complex denaturing reaction, and makes the renaturation reaction continue, is carried out with high protein renaturation concentration, is convenient for removing a denaturing agent, integrates renaturation and purification processes, and can realize automation and scale, and a chromatographic medium can be reused, which reduces the process cost and is more conducive to industrialized massproduction in the future.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically, relates to a method for preparing protein inclusion body and recombinant human beta-nerve growth factor. Background technique [0002] Nerve growth factor (NGF) is the earliest discovered and most thoroughly studied neurotrophic factor that has the function of nourishing neuron cell bodies and promoting the growth of neuron axons. Animal experiments and clinical studies have shown that based on its regulation of central and peripheral nerves After nerve injury, exogenous administration of NGF can promote the repair and regeneration of damaged nerves, so it is a potential drug for the treatment of nerve injury-related diseases. NGF is a complex protein consisting of three subunits, α, β, and γ, among which the β subunit has the function of nutrition and promoting nerve growth, which is formed by non-covalent combination of two single peptide chains composed of 118 amino acids T...

Claims

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Application Information

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IPC IPC(8): C12P21/06C07K14/48C07K1/113C07K1/16
CPCC12P21/06C07K14/48
Inventor 李树刚张伟王传胜邓永康龚会英柴新娟但国平程丹凝余英鹏
Owner 重庆科润生物医药研发有限公司
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