Circulating operating method which can be independently used for protein renaturation or used as guiding operation of protein renaturation

A protein and leading technology, applied in the field of basic biology, can solve the problem of inability to get rid of tail fluid pollution, achieve easy recovery and reuse, improve renaturation efficiency, and reduce solution volume.

Active Publication Date: 2016-12-21
张鹏
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  • Abstract
  • Description
  • Claims
  • Application Information

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  • Circulating operating method which can be independently used for protein renaturation or used as guiding operation of protein renaturation
  • Circulating operating method which can be independently used for protein renaturation or used as guiding operation of protein renaturation
  • Circulating operating method which can be independently used for protein renaturation or used as guiding operation of protein renaturation

Examples

Experimental program
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Effect test

Embodiment 1

[0052] First, 250 milliliters of urea solution with a concentration of 8 mol / liter and 50 microliters of dimercaptoethanol are mixed for the first treatment to obtain a denaturing solution; take 200 milliliters of the obtained denaturing solution and 20 mg of crushed EGF inclusion bodies Carry out the second mixed treatment to obtain the inclusion body solution whose concentration is 0.1mg / ml; add 50 grams of 25wt% sucrose and 20 milliliters of 10wt% glycerin to the obtained inclusion body solution, and utilize sodium carbonate solution to denature the solution Adjust the pH to 7-8; place the obtained mixed solution at minus 20 degrees Celsius for program cooling. When a large amount of urea crystals are precipitated, fish them out, and continue to freeze the rest until the ice cubes come out. There is obviously a boundary area in the urea crystallization. Take out the ice cubes on the top and store them in a centrifuge tube at minus 20 degrees Celsius to separate and obtain th...

Embodiment 2

[0057]First, 250 milliliters of urea solution with a concentration of 8 mol / liter is mixed with 0.2 milliliters of dimercaptoethanol to obtain a denatured solution; 1.4 liters of the obtained denatured solution is mixed with 140 mg of crushed EGF inclusion bodies In the second mixed treatment, the inclusion body solution with a concentration of 0.1 mg / ml is obtained; 100 ml of glycerin is added as an antifreeze protection agent in the obtained inclusion body solution, and the freezing point of the solution is adjusted, and the pH of the denaturing solution is adjusted to 7.1; Wrap the container with several layers of towels to keep warm, place the obtained mixed solution at minus 20 degrees Celsius for programmed cooling, observe the precipitation situation, and remove excessive crystals. After the surface liquid freezes, extract the floating ice layer and melt the obtained protein After that, the refolded protein can be obtained, and then 3 samples of the obtained refolded pro...

Embodiment 3

[0061] First, 250 milliliters of urea solution with a concentration of 8 mol / liter is mixed with 0.2 milliliters of dimercaptoethanol to obtain a denatured solution; 1.4 liters of the obtained denatured solution is mixed with 140 mg of crushed EGF inclusion bodies In the second mixing treatment, an inclusion body solution with a concentration of 0.1 mg / ml was obtained; 100 ml of glycerin was added to the obtained inclusion body solution as an antifreeze protection agent to adjust the freezing point of the solution, and the pH of the denatured solution was adjusted to 7.1 by using sodium carbonate solution Wrap the container with several layers of towels for heat preservation, then place the obtained mixed solution at minus 14 degrees Celsius for programmed cooling, and directly pour out a large amount of concentrated solution, which contains the quasi-refolding precursor of EGF. This is the leading operation, and the obtained solution can be easily connected with other renatura...

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Abstract

The invention discloses a circulating operation method which can be independently used for protein renaturation or used as guiding operation of protein renaturation. The method comprises the following steps: (1) adding a sample to be subjected to renaturation into a denaturation liquor for dissolving; (2) gradually separating out a denaturing agent from the liquor in a low-temperature environment, wherein in a micro level, denaturation salinity smooth decent gradient is generated at the molecular level; and gradually forming a renaturation and/or quasi-renaturation state protein precursor in the liquor, wherein along with separation by crystallization of the denaturing agent, the volume of the liquor is reduced, and the protein concentration is greatly increased; (3) separating a renaturation and/or quasi-renaturation state protein precursor; (4) dissolving crystal of the denaturing agent separated out in step (2) and the solution of the renaturation protein and/or quasi-renaturation state protein precursor separated out in step (3), and returning to the state of step (1), thus completing one production cycle. Various working media can be flexibly adjusted according to conditions. The method can be used for efficiently preparing a renaturation and/or quasi-renaturation state protein precursor.

Description

technical field [0001] The invention belongs to the field of basic biological technology, in particular, the invention relates to a cyclic operation method which can be used independently for protein renaturation or as a leading operation of protein renaturation. Background technique [0002] At present, the world's annual recombinant protein product market exceeds US$160 billion (1H.-P.Meyer, D. Schmidhalter in Innovations in Biotechnology (Ed.: E.Agbo), InTech, Rijeka, 2012, pp.211–250.), how Efficient and cheap production of protein products is a major project pursued by scientific research and industry. [0003] Since the 1930s, pioneers in scientific research have discovered that some denatured proteins can regain biological activity by slowly reducing the concentration of denaturant. In the 1980s, with the gradual maturity of PCR technology, the research on renaturation of inclusion bodies produced by extremely cheap and efficient Escherichia coli once became the focu...

Claims

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Application Information

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IPC IPC(8): C07K1/14
Inventor 张鹏
Owner 张鹏
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