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Method for assisting lysozyme in vitro refolding by means of linear poly N-isopropyl acrylamide

A technology of isopropylacrylamide and lysosome, which is applied in the direction of biochemical equipment and methods, enzymes, enzymes, etc., can solve the problems of protein loss, cumbersome preparation steps, insoluble, etc., achieve fewer parameters, inhibit aggregation tendency, Easy to control and amplify effects

Inactive Publication Date: 2008-11-05
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the preparation steps of this method are cumbersome, and the obtained microspheres are limited to assisting in vitro renaturation of proteins containing more sulfhydryl groups or disulfide bonds.
Our research group once synthesized granular poly-N-isopropylacrylamide and investigated its effect of assisting lysozyme refolding (see the method of temperature-sensitive poly-N-isopropylacrylamide gel assisting lysozyme refolding, invention patent Application number: ZL 2004 1 0073406.9, date of authorization: August 2, 2006), the polymer is a cross-linked network gel, which can be significantly swelled in water, but cannot be dissolved, because the surface of the granular gel will absorb some protein, resulting in protein loss

Method used

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  • Method for assisting lysozyme in vitro refolding by means of linear poly N-isopropyl acrylamide
  • Method for assisting lysozyme in vitro refolding by means of linear poly N-isopropyl acrylamide
  • Method for assisting lysozyme in vitro refolding by means of linear poly N-isopropyl acrylamide

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Experimental program
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Effect test

Embodiment 1

[0018] 1) Dissolve N-isopropylacrylamide and azobisisobutyronitrile with a molar concentration ratio of 10:1 in absolute ethanol. The concentration of N-isopropylacrylamide is 1mol / L, and it is protected under nitrogen at 60℃. After reacting for 12 hours and evaporating under reduced pressure to remove most of the ethanol, the resulting reaction product was dissolved in acetone, and the dissolved solution was slowly dropped into the constantly stirring n-hexane to obtain a white precipitate. The reaction product dissolution and precipitation steps were repeated 3 times to obtain a linear polymer N-isopropyl acrylamide, dried in a vacuum dryer to constant weight. The number average molecular weight of the resulting linear poly-N-isopropylacrylamide is M n =20.4kDa, the weight average molecular weight is M w = 30.8 kDa, polydispersity = 1.51.

[0019] 2) Dissolve lysozyme in denaturation buffer to prepare a solution with a concentration of 10 mg / mL, and place the solution in a shake...

Embodiment 2

[0021] 1) Dissolve N-isopropylacrylamide and azobisisobutyronitrile with a molar concentration ratio of 10:1 in absolute ethanol. The concentration of N-isopropylacrylamide is 1mol / L, and it is protected under nitrogen at 60℃. After reacting for 12 hours and evaporating under reduced pressure to remove most of the ethanol, the resulting reaction product was dissolved in acetone, and the dissolved solution was slowly dropped into the constantly stirring n-hexane to obtain a white precipitate. The reaction product dissolution and precipitation steps were repeated 3 times to obtain a linear polymer N-isopropyl acrylamide, dried in a vacuum dryer to constant weight. The number average molecular weight of the resulting linear poly-N-isopropylacrylamide is M n =20.4kDa, the weight average molecular weight is M w = 30.8 kDa, polydispersity = 1.51.

[0022] 2) Dissolve lysozyme in denaturation buffer to prepare a solution with a concentration of 10 mg / mL, and place the solution in a shake...

Embodiment 3

[0024] 1) Dissolve N-isopropylacrylamide and azobisisobutyronitrile with a molar concentration ratio of 10:1 in absolute ethanol. The concentration of N-isopropylacrylamide is 1mol / L, and it is protected under nitrogen at 60℃. After reacting for 12 hours and evaporating under reduced pressure to remove most of the ethanol, the resulting reaction product was dissolved in acetone, and the dissolved solution was slowly dropped into the constantly stirring n-hexane to obtain a white precipitate. The reaction product dissolution and precipitation steps were repeated 3 times to obtain a linear polymer N-isopropyl acrylamide, dried in a vacuum dryer to constant weight. The number average molecular weight of the resulting linear poly-N-isopropylacrylamide is M n =20.4kDa, the weight average molecular weight is M w = 30.8 kDa, polydispersity = 1.51.

[0025] 2) Dissolve lysozyme in denaturation buffer to prepare a solution with a concentration of 10 mg / mL, and place the solution in a shake...

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Abstract

The invention discloses a method adopting linear poly N-isopropyl acrylamide adhesive to assist the renaturation of muramidase in vitro, comprising the following steps that: 1) the linear N-isopropyl acrylamide is prepared by a free radical polymerization method; 2) the linear poly N-isopropyl acrylamide is used to assist the renaturation of muramidase in vitro. The linear poly N-isopropyl acrylamide is taken as a novel protein in vitro additive which can increase the processing concentration of the renaturation muramidase, lower the production cost, and improve the activity recovery rate of the muramidase up to about 80 percent when the processing concentration is 600 Mug / mL; while the activity recovery rate of the dilution renaturation on the same condition is only 10 percent. The gel has the advantages of high efficiency, convenient operation and simple technique, etc., thereby having good potential application in the protein in vitro renaturation field.

Description

Technical field [0001] The invention relates to a method for assisting the renaturation of lysozyme in vitro by linear poly-N-isopropylacrylamide. Background technique [0002] In the process of industrialization of genetically engineered proteins, there is often such a problem: recombinant expression products usually exist in the form of non-biologically active aggregates, that is, inclusion bodies. How to refold the protein efficiently into a protein with natural conformation and natural activity is the key to determining whether the genetically engineered protein can be industrialized and used by humans. This makes the development of protein in vitro renaturation technology a recent trend. One of the research hotspots. However, due to the diversity of proteins, the structure and the complexity of the folding process, people cannot simply transplant a renaturation method, and it is difficult to find a universal and low-cost efficient renaturation method. Therefore, in the actua...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/36
Inventor 关怡新陈莹莹葛翔姚善泾
Owner ZHEJIANG UNIV
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