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Method for assisting lysozyme renaturation by temperature sensitive type poly N-isopropyl acrylamide gel

An isopropyl acrylamide gel, lysozyme renaturation technology, applied in biochemical equipment and methods, enzymes, immobilized on/in organic carriers, etc., can solve problems such as troublesome operation and protein loss

Inactive Publication Date: 2005-08-03
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, when the linear polymer is used as an additive to assist renaturation, the liquid PNIPA must be precipitated from the aqueous solution by heating after renaturation, and then a step of centrifugation can be used to achieve the separation of the target protein and the PNIPA water-soluble polymer after renaturation. And it is easy to cause the loss of protein near the interface between the polymer and the protein aqueous solution, and the operation is relatively troublesome
So far, there are no related reports about granular PNIPA gel assisting protein renaturation in vitro

Method used

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  • Method for assisting lysozyme renaturation by temperature sensitive type poly N-isopropyl acrylamide gel
  • Method for assisting lysozyme renaturation by temperature sensitive type poly N-isopropyl acrylamide gel

Examples

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Effect test

Embodiment 1

[0020] Embodiment 1: the preparation of thermosensitive type PNIPA gel (total monomer concentration 10%, cross-linking degree 5%)

[0021] The reaction was carried out in a 250mL three-necked bottle, and the stirring speed was adjusted to 420rpm by an electronic constant speed stirrer. The whole reactor is submerged in a super constant temperature tank, and the polymerization temperature is 25°C. Dissolve 75mg of surfactant Tween80 in 75mL of liquid paraffin in the continuous phase, add it to the reactor, and feed an appropriate flow rate of N 2 , stirred for about 20 minutes to mix the solution evenly, then slowly added the aqueous solution of the reactant with a total monomer concentration of 10% and a cross-linking degree of 5% into the reactor dropwise with a pipette gun, continued to stir for 30 minutes, and then slowly added 1 μL initiator TEMED 20 μL, polymerize for 3 hours and discharge. The final product is repeatedly soaked with different volume ratios of absolute ...

Embodiment 2

[0024] Embodiment 2: the preparation of granular PNIPA gel (total monomer concentration 14%, degree of cross-linking 10%)

[0025] The reaction was carried out in a 250mL three-necked bottle, and the stirring speed was adjusted to 420rpm by an electronic constant speed stirrer. The whole reactor is submerged in a super constant temperature tank, and the polymerization temperature is 25°C. Dissolve 75mg of surfactant Tween80 in the continuous phase, add to the reactor, and feed into the N of appropriate flow rate 2 , stirred for about 20 minutes to mix the solution evenly, and then slowly added the aqueous solution of the reactant with a total monomer concentration of 14% and a cross-linking degree of 10% into the reactor dropwise with a pipette gun, continued to stir for 30 minutes, and then slowly added 1 μL initiator TEMED, polymerized at 25°C for 3 hours and discharged. The final product is repeatedly soaked with different volume ratios of absolute ethanol / water solution,...

Embodiment 3

[0027] Embodiment 3: granular PNIPA gel assists lysozyme refolding (10% of the total monomer concentration of preparation gel, cross-linking degree 5%)

[0028] After adding 10mg / mL inactive lysozyme solution in the extended state to the refolding buffer (the dilution factor is 40), add 100mg / mL granular PNIPA gel to it, fully shake and disperse evenly, then shake at 30°C and 120rpm Refolding in bed for 3 hours, the results are as follows figure 1 shown. After renaturation, the activity recovery rate of lysozyme reached 50.34%, but under the same conditions, the recovery rate of activity was only 44.9%.

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Abstract

The method for assisting lysozyme renaturation with temperature-sensitive poly N-isopropyl acrylamide gel includes the following steps: 1) compounding poly N-isopropyl acrylamide gel aqua; 2) preparing poly N-isopropyl acrylamide gel in reverse suspension process; and 3) assisting lysozyme renaturation with temperature-sensitive poly N-isopropyl acrylamide gel. The temperature-sensitive poly N-isopropyl acrylamide gel the present invention develops as one novel additive for extracorporeal renaturation of protein can raise the lysozyme activity recovering rate. The gel has high efficiency, less renaturation affecting parameters, convenient operation, relax requirement in instrument and apparatus, easy separation and recovery and other advantages. The present invention has excellent application foreground in extracorporeal renaturation of protein.

Description

technical field [0001] The invention relates to a method for assisting lysozyme refolding by thermosensitive poly N-isopropylacrylamide gel. Background technique [0002] The industrialization of genetic engineering products often faces such a problem: the expressed products usually exist in the form of aggregates without biological activity. How to efficiently refold protein into an active protein with natural conformation is the key to determine whether the genetically engineered protein can be industrialized and utilized by humans. This makes the research on protein in vitro renaturation technology become a research hotspot in recent years. However, due to the diversity of proteins, the complexity of structure and 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 actual operation process, it is necessary to optimize each factor affecting renaturati...

Claims

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Application Information

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IPC IPC(8): C12N9/36C12N11/04
Inventor 关怡新姚善泾崔志芳
Owner ZHEJIANG UNIV
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