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Method for separating and purifying protein through aqueous-two-phase system

A two-phase system, separation and purification technology, applied in peptide preparation methods, chemical instruments and methods, animal/human proteins, etc., can solve the problems of low efficiency of protein back extraction, difficult separation and purification of proteins, weak phase separation ability of DES, etc. problems, achieve protein structure and activity retention, high stripping efficiency, and low cost

Active Publication Date: 2019-07-12
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are some problems when the DES-inorganic salt two-phase system is applied to the separation and purification of proteins: (1) It is difficult to further separate and purify proteins from the DES phase after extraction, and the back extraction efficiency of proteins is low, although proteins and DES can be separated by dialysis , but the speed is slow and the water consumption is large; (2) The phase separation ability of DES is weak, and a high salt concentration is required to form a two-phase system; (3) The DES phase is located in the upper layer after centrifugation, which is inconvenient to collect

Method used

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  • Method for separating and purifying protein through aqueous-two-phase system
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  • Method for separating and purifying protein through aqueous-two-phase system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] The effect of deep eutectic solvent type and molar ratio on the extraction rate of two-phase system:

[0052] Aqueous two-phase extraction: Weigh 1.6 g of DES into a 5 mL centrifuge tube, add 2 mL of 0.03 g / mL K containing 10 mg bovine serum albumin (BSA) 2 HPO 4 aqueous solution. Shake at 1000rpm for 10min at 25°C or vortex at 60 W for 10min, and centrifuge at 3000rpm for 5min. At this time, a mutually incompatible liquid-liquid two-phase system is formed, in which the lower layer is the DES phase and the upper layer is the salt phase. Record the volume of the two phases, collect the DES phase and dilute it 20 times with water for UV measurement, take the reference substance comparison method to quantitatively analyze the content of BSA, and calculate the extraction rate. The results are shown in Table 1.

[0053] It can be seen from Table 1 that both ChCl-HFIP DES and tetramethylammonium chloride-HFIP DES have higher protein extraction rates. The extraction rates...

Embodiment 2

[0057] The influence of different extraction conditions on the extraction rate of the two-phase system:

[0058] Aqueous two-phase extraction: Weigh ChCl-HFIP DES with a certain mass molar ratio of 1:2 and place it in a 5mL centrifuge tube, add 2mL of a certain concentration of K containing a certain mass of BSA 2 HPO 4 aqueous solution. Shake at 1000 rpm for a certain time at a certain temperature, and centrifuge at 3000 rpm for 5 minutes. At this time, a mutually incompatible liquid-liquid two-phase system is formed, in which the lower layer is the DES phase and the upper layer is the salt phase. Record the volume of the two phases, collect the DES phase and dilute it with water 15-200 times for UV measurement, take the reference substance comparison method to quantitatively analyze the content of BSA, and calculate the extraction rate. The results are shown in Table 2.

[0059] Extraction rate under different extraction conditions in table 2

[0060]

[0061]

[...

Embodiment 3

[0064] The influence of different stripping conditions on the stripping rate:

[0065] 1. Aqueous two-phase extraction:

[0066] Weigh 1.33 g of ChCl-HFIP DES with a molar ratio of 1:2 and place it in a 5 mL centrifuge tube, add 2 mL of 0.022 g / mL K containing 5 mg BSA 2 HPO 4 aqueous solution. Shake at 1000rpm for 5min at 25°C and centrifuge at 3000rpm for 5min. At this time, a mutually incompatible liquid-liquid two-phase system is formed, in which the lower layer is the DES phase and the upper layer is the salt phase. Record the volume of the two phases, collect the DES phase and dilute it 20 times with water for UV measurement, and use the reference substance comparison method to quantitatively analyze the content of BSA. The extraction rate is 99.20%-100.00%, and almost all of the BSA is extracted into the DES phase.

[0067] 2. Back extraction protein:

[0068] The DES phase after protein extraction was placed in a 5 mL centrifuge tube, diluted 7.5, 8, 9, 10, 11, a...

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Abstract

The invention relates to the technical field of separation and purification of natural products, in particular to a method for separating and purifying protein through an aqueous-two-phase system. Themethod comprises the following steps of A, protein extraction through the aqueous-two-phase system, wherein a low-eutectic solvent and an inorganic salt aqueous solution containing the protein are uniformly mixed, then centrifugation is conducted to obtain a liquid-liquid two-phase system, and then a hypophase extraction liquid is collected; B, protein reextraction, wherein the hypophase extraction liquid in step A is diluted with water and then centrifuged to obtain a liquid-solid two-phase system; C, preparation of a pure product of the protein, wherein a solid phase in step B is dissolvedwith water, and freeze drying is conducted after ultrafiltration centrifugation to obtain the pure product of the protein, wherein in step A, the low-eutectic solvent is prepared from quaternary ammonium salt and hexafluoroisopropanol. According to the method, a DES-salt aqueous-two-phase system based on HFIP is adopted, the DES phase is easy to collect, the extraction rate and reextraction rate on the protein are high, the protein purity is high, and the structure and activity of the protein are both maintained; the operation is simple.

Description

technical field [0001] The invention relates to the technical field of separation and purification of natural products, in particular to a method for separation and purification of proteins in a two-phase aqueous system. Background technique [0002] Protein is the material basis of life activities, and its separation and purification have attracted more and more attention. Traditional protein purification methods include ammonium sulfate precipitation, ion exchange chromatography, affinity chromatography, gel filtration chromatography, etc., which have problems such as cumbersome operation, long time-consuming, and high cost. Therefore, it is crucial to develop fast, simple and effective protein purification techniques. [0003] Aqueous two-phase system (ATPS) is a water solution of one or several substances spontaneously formed under certain conditions, two mutually immiscible, clear interface aqueous phase system, based on the selective distribution of analytes in the tw...

Claims

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Application Information

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IPC IPC(8): C07K14/765C07K1/14
CPCC07K14/765
Inventor 肖玉秀王璇璇
Owner WUHAN UNIV
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