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Method for inclusion body protein dilution and dialysis renaturation

An inclusion body and protein technology, applied in the field of inclusion body protein dilution dialysis refolding, can solve the problems of incorrectly folded protein configuration, low protein recovery rate, re-aggregation, etc., to promote protein refolding, high concentration, and reduce additives effect of concentration

Active Publication Date: 2019-06-28
安徽环球基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The yield of the first two steps is relatively high, and the yield of the third step is low, and the incorrectly folded protein configuration is the main limiting factor
[0004] At present, there are mainly two methods for inclusion body renaturation: dilution renaturation and dialysis, ultrafiltration or electroosmosis to remove denaturants; among them, dilution renaturation and dialysis renaturation are the simplest and are widely used in laboratories for protein function and Structural research, but in the renaturation process, it is easy to cause protein degradation or re-aggregation, making the protein recovery rate low

Method used

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Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0033] The second step, the preparation of composite membrane

[0034] Dissolve 0.5g of modified chitosan prepared in the first step in 200ml of DMF, add 20g of polymethacrylate and 0.2g of polyethylene glycol (molecular weight is 1000), after the solution is mixed evenly, put it into a shaker for 2 day, the temperature of the shaker was controlled at 40°C, and the rotation speed was 120r / min. After the shaking was completed, vacuum defoaming was carried out at a vacuum degree of 0.05MPa, and then evenly cast on a smooth glass plate, scraped, and at room temperature Place it for 4-6 hours, put it into deionized water with a temperature of 20°C to form a gel film, soak it in deionized water for 20 hours, take it out and put it in a blast drying oven for 9 hours at 60°C to obtain the modified chitosan-polymer Methacrylate composite film;

[0035] Described polymethacrylate is a kind of in polymethyl methacrylate, polyethyl methacrylate;

[0036] The infrared characterization o...

Embodiment 1

[0038] A method for diluting dialysis renaturation of inclusion body protein, specifically comprising the following steps:

[0039] 1) Dissolving the inclusion body protein to be used with a denaturant to obtain the inclusion body protein with a solubility of 8 mg / ml;

[0040] 2) At 4°C, slowly add the inclusion body protein dissolved in step 1) dropwise to the refolding buffer to dilute it 8 times, stir while adding, and let stand at 4°C for 12 hours after the dropwise addition , the concentration of inclusion body protein is 1 mg / ml at this time;

[0041] 3) Centrifuge the solution obtained in step 2) at 16,000 rpm for 10 minutes, collect the protein supernatant, and dilute the protein supernatant with a basic buffer at 4°C; the basic buffer and the protein supernatant are The volume ratio is 1:1;

[0042] 4) centrifuge the protein supernatant diluted in step 3) at 16000rpm for 10min, collect the supernatant, and dialyze the collected supernatant into the base buffer at 4°...

Embodiment 2

[0048] A method for diluting dialysis renaturation of inclusion body protein, specifically comprising the following steps:

[0049] 1) Dissolving the inclusion body protein to be used with a denaturant to obtain the inclusion body protein with a solubility of 20 mg / ml;

[0050] 2) At 4°C, slowly add the inclusion body protein dissolved in step 1) dropwise to the refolding buffer to dilute it 8 times, stir while adding, and let stand at 4°C for 12 hours after the dropwise addition , the concentration of inclusion body protein is 2 mg / ml at this time;

[0051] 3) Centrifuge the solution obtained in step 2) at 16,000 rpm for 10 minutes, collect the protein supernatant, and dilute the protein supernatant with a basic buffer at 4°C; the basic buffer and the protein supernatant are The volume ratio is 1:1;

[0052] 4) centrifuge the protein supernatant diluted in step 3) at 16000rpm for 10min, collect the supernatant, and dialyze the collected supernatant into the base buffer at 4...

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Abstract

The invention discloses a method for inclusion body protein dilution and dialysis renaturation. A mild degeneration method is adopted for dissolving inclusion body protein, an existing secondary structure in the inclusion body protein is protected against damage so that inclusion body protein renaturation is greatly facilitated; the method integrates dilution and dialysis for performing protein renaturation, the concentration of a degeneration agent is lowered by first-time dilution, protein renaturation is promoted, the concentration of an additive is lowered by second-time dilution, the protein is more easily suitable for the environment of a basic buffer solution, finally, dialysis is performed to remove various additives, and the renaturated protein with the high renaturation efficiency and the high concentration can be obtained.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for dilution dialysis refolding of inclusion body protein. Background technique [0002] With the rapid development of the genetic engineering application industry, it is possible to express and obtain biologically active proteins in vitro and apply them to large-scale factories. Escherichia coli is often used as a host cell for the expression of recombinant proteins because of its easy handling, clear genetic background, low production cost and high expression level. High-efficiency protein expression in prokaryotic host cells often aggregates to form high-density, insoluble protein particles wrapped by membranes, that is, inclusion bodies. Inclusion bodies are aggregates of randomly stretched peptide chains, without biological activity, and inclusion body proteins need to be dissolved and then refolded. [0003] So far, there is no renaturation scheme universally applica...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/14C07K1/34
Inventor 雍金贵张伦
Owner 安徽环球基因科技有限公司
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