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Renaturation method of restructured human insulin prokaryotic-fusion protein

A technology of recombinant human insulin and fusion protein, applied in the field of protein renaturation, can solve the problems of low renaturation rate, low protein concentration, complicated production process, etc., and achieve the effects of improving renaturation rate, simple operation and simple processing method.

Active Publication Date: 2016-07-06
TONGHUA DONGBAO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] Therefore, the technical problem to be solved by the present invention is to provide a method for renaturation of Escherichia coli expressing recombinant human proinsulin fusion protein suitable for industrial production, so as to overcome the low protein concentration during renaturation and the production process in the prior art. Complex technical defects with low refolding rate

Method used

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  • Renaturation method of restructured human insulin prokaryotic-fusion protein
  • Renaturation method of restructured human insulin prokaryotic-fusion protein
  • Renaturation method of restructured human insulin prokaryotic-fusion protein

Examples

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Effect test

Embodiment 1

[0040] Preparation of recombinant human proinsulin fusion protein

[0041] Step 1: Collection of recombinant human proinsulin fusion protein inclusion bodies

[0042] After fermentation by Escherichia coli, wash twice with purified water, determine the weight, add 100mL of purified water for every 10g of bacteria, mix well, crush in a homogenizer at 900-950bar, after the bacteria are completely broken, use a centrifuge Centrifuge at 12000g for 10 minutes to collect the precipitate. The obtained precipitate is mainly inclusion bodies. Wash twice according to the following method to remove most of the non-recombinant human proinsulin fusion protein: use 100mL washing buffer for every 10g of precipitate, containing 50mM Tris-HCl (pH9 .0), 2mM EDTA, 1% Triton-X100 (mass ratio), 3M urea. Stir and mix at room temperature, let stand for 30 minutes, and centrifuge at 12,000 g for 10 minutes, so as to collect the precipitate twice.

[0043]Step 2: Dissolution and reduction of recombi...

Embodiment 2

[0048] Preparation of recombinant human proinsulin fusion protein

[0049] The recombinant human proinsulin fusion protein after denaturation and reduction, in the refolding buffer, the protein concentration reaches 7.5mg / mL, and the concentration of other components is: urea with a final concentration of 0.8M, oxidized glutathione with a final concentration of 1mM Glycine, reduced glutathione with a final concentration of 1mM, EDTA with a final concentration of 2mM, and β-mercaptoethanol with a final concentration of 2mM were adjusted to pH 9.0-9.5, and placed at 4°C. It can be observed that in the refolding solution, the solution remains transparent and no precipitation occurs. Other operations are the same as in Example 1. The calculated renaturation rate of recombinant human proinsulin fusion protein was 85.34%.

Embodiment 3

[0051] Preparation of recombinant human proinsulin fusion protein

[0052] The recombinant human proinsulin fusion protein after denaturation and reduction, in the refolding buffer, the protein concentration reaches 10mg / mL, and the concentration of other components is: urea with a final concentration of 0.8M, and oxidized glutathione with a final concentration of 5mM Peptide, reduced glutathione at a final concentration of 1 mM, EDTA at a final concentration of 2 mM, and β-mercaptoethanol at a final concentration of 3 mM, adjusted to pH 9.0-9.5, and placed at 4°C. It can be observed that in the refolding solution, the solution remains transparent and no precipitation occurs. Other operations are the same as in Example 1. The calculated refolding rate of recombinant human proinsulin fusion protein was 82.59%.

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Abstract

The invention relates to a renaturation method of restructured human insulin prokaryotic-fusion protein and belongs to the field of protein purification. The renaturation method includes steps of 1) smashing escherichia coli expressing restructured human insulin prokaryotic-fusion protein, and collecting inclusion bodies; 2) washing the inclusion bodies, dissolving the inclusion bodies and modifying the fusion protein; 3) renaturating protein. The renaturation method is needless of protein purification in advance, directly performs protein renaturation, and finally obtains high-concentration restructured insulin prokaryotic-fusion protein of natural structure. By the renaturation method, the defect of low protein concentration after protein renaturation and resultantly easy protein accumulation and precipitation is overcome, renaturation efficiency is up to 80-90%, production efficiency is improved, and the renaturation method is applicable to industrial production.

Description

technical field [0001] The invention belongs to a protein refolding method, in particular to a refolding method for recombinant human proinsulin fusion protein. Background technique [0002] Diabetes mellitus is a metabolic disorder characterized by chronic hyperglycemia caused by various etiologies. It is a syndrome caused by the absolute or relative lack of insulin in the body or the insensitivity of target tissues to insulin. In recent years, with the improvement of people's living standards, the incidence of diabetes has shown an increasing trend year by year, becoming the third leading cause of death after cardiovascular and tumors. According to the data released by the International Diabetes Federation (IDF), in 2014 The number of people with diabetes worldwide (aged 20 to 79) reached 387 million, an increase of 5 million from last year, and is expected to reach 592 million in 2035. A large number of clinical practices have confirmed that the treatment of diabetes wit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K1/14C07K1/113
CPCC07K14/62C07K2319/00
Inventor 冷春生李一奎魏宏壮常晓慧
Owner TONGHUA DONGBAO PHARMA
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