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Method for renaturation of protein

A protein and denaturant technology, applied in the field of renatured proteins, can solve the problems of unsuitability for large-scale production, decreased activity recovery, and the volume cannot be too large, and achieves the effects of convenient large-scale application, simple operation, and reduced difficulty.

Inactive Publication Date: 2005-09-21
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the gel filtration method is the most widely used, but it has similar disadvantages to the separation process, that is, the sample volume cannot be too large, so the processing volume is small, and it is also not suitable for large-scale production.
Due to the principle of charge adsorption, the ion exchange method can adsorb a large amount of protein, but as the concentration of denaturant in the chromatography column decreases, the denatured protein adjacent to the adsorption will also aggregate, resulting in a decrease in activity recovery
While other methods are still very immature, only a few proteins have been exploratoryly studied at low concentrations

Method used

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  • Method for renaturation of protein
  • Method for renaturation of protein
  • Method for renaturation of protein

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Renaturation of Denatured Lysozyme on Poros PE 50 Hydrophobic Chromatographic Column Eluted with Glycerol

[0026] Perfuse Poros PE 50 hydrophobic chromatography column (128mm×10mm I.D, 10mL) with mobile phase I (3.6M (NH 4 ) 2 SO 4 , 50mM Tris-HCl, 1mM EDTA, 3mM GSH, 0.3mM GSSG, pH8.5) equilibrated, run mobile phase I ~ denaturant A (8M urea, 50mM Tris-HCl, 1mM EDTA, 3mM GSH, 0.3mM GSSG, pH8.5) gradient 2ml, dissolving 10mg of lysozyme at a concentration of 50mg / ml with denaturant B (6M guanidine hydrochloride, 50mM Tris-HCl, 1mMEDTA, 1% 2-mercaptoethanol, pH8.5) and washing with mobile phase I to adsorb the denatured protein , and then change the eluent of mobile phase II and osmolyte (50% glycerol, 0.4M (NH 4 ) 2 SO 4 , 100mM Tris-HCL, 1mM EDTA, 0.1% 2-mercaptoethanol, pH8.5) to wash one column volume, and then run mobile phase II to denaturant C (8M urea, 50mM Tris-HCl, 1mM EDTA, 0.1% 2-mercaptoethanol, pH8.5) gradient 4ml and denaturant C ~ mobile phase II gr...

Embodiment 2

[0033] Refolding of recombinant human lysozyme inclusion body solution on Poros PE 50 hydrophobic chromatography column eluted with glycerol

[0034] A Poros PE 50 chromatography column (128mm×10mm I.D, 10mL) was used with mobile phase I (3.6M (NH 4 ) 2 SO 4 , 50mMTris-HCl, 1mM EDTA, 0.1% 2-mercaptoethanol, pH8.5) equilibrated, run the mobile phase I ~ denaturant A (8M urea, 50mMTris-HCl, 1mMEDTA, 3mMGSH, 0.3mM GSSG, pH8.5) gradient 2ml, containing denaturant B (6M guanidine hydrochloride, 50mM Tris-HCl, 1mM EDTA, 1% 2-mercaptoethanol, pH8.5) human lysozyme solution 2mg, wash with mobile phase I, let the denatured protein adsorb, and then change the flow Phase II (50% glycerol, 0.4M (NH 4 ) 2 SO 4 , 100mM Tris-HCL, 1mM EDTA, 0.1% 2-mercaptoethanol, pH8.5) to wash one column volume, and then run mobile phase II to denaturant C (8M urea, 50mM Tris-HCl, 1mM EDTA, 0.1% 2-mercaptoethanol , pH8.5) gradient 5ml and denaturant C ~ mobile phase II gradient 5ml, continue to elute ...

Embodiment 3

[0038] Refolding of Recombinant Human α-2b Interferon on Phenyl Sepharose FF Hydrophobic Chromatography Column Eluted with Mannitol

[0039] Equilibrate the Phenyl Sepharose FF chromatography column (25mm×16mm I.D, 5mL) with mobile phase I (0.1N acetic acid), and denature it with 6M guanidine hydrochloride, 50mM Tris-HCl, 1mMEDTA, 1% 2-mercaptoethanol, pH8.5 The reduced recombinant α-2b interferon inclusion body was washed with mobile phase I to absorb the denatured protein, and then changed to mobile phase II (20% mannitol, 0.2M NaCl, 20mM Na 2 HPO 4 , 0.01% 2-mercaptoethanol) to wash a column volume, and then run mobile phase II ~ denaturant C (8M urea, 10mM Na 2 HPO 4 , 0.01% 2-mercaptoethanol) gradient 5ml and denaturant C ~ mobile phase II gradient 5ml, continue to elute with mobile phase II, the flow rate is 2ml / min, and the specific activity of α-2b interferon eluted is 1.5× 10 8 IU / mg, the total activity yield is up to 80%.

[0040] It shows that the present inven...

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Abstract

The present invention relates to a method for renaturating protein by adopting penetrating matter and hydrophobic chromatography combination system. Its basic process includes the following steps: adopting high-concentration salt and denaturant to gradient adsorb the denatured protein on the hydrophobic chromatographic column, then using penetrating matter with a certain concentration to make elution so as to make the protein progressively be renaturated in the course of elution, after correct folding, can obtain the natural activity protein. Said invention can obtain active protein with high concentration, and can retain high protein recovery and activity recovery.

Description

technical field [0001] The invention relates to a method for refolding protein, in particular to a new method for refolding inclusion body protein or denatured natural protein by using a combined system of osmolyte and hydrophobic chromatography. Background technique [0002] At present, biotechnology has been developed rapidly, and one of the important fields is to use gene recombination technology to mass-produce nature and its trace proteins. Escherichia coli and other prokaryotic organisms are usually the preferred host bacteria for gene recombination technology due to their fast growth rate and low nutritional requirements. However, foreign proteins highly expressed in Escherichia coli often form insoluble and inactive inclusion bodies, which require people to first dissolve the inclusion bodies with a denaturant, and then remove the denaturant to restore its natural activity. Known as protein folding renaturation. There is an irreconcilable contradiction in protein f...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/00
Inventor 苏志国李京京
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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