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Method of separating protein aggregate in mixed liquid of denatured protein/incusion protein renaturation

A technology for protein aggregates and protein refolding, which is applied in the field of separation of protein aggregates in the denatured protein/inclusion body protein refolding mixture, which can solve the problems of inability to separate components, complex protein components, and difficult separation of various components

Inactive Publication Date: 2005-06-22
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In the literature on protein renaturation, although the formation of aggregates is a recognized difficulty in renaturation and a hot spot in renaturation research, there are few reports on the separation of proteins after renaturation
The reason is that the composition of the protein after refolding is quite complex, and there are various forces between the protein molecules of various structures and between the protein molecules and the separation medium, and the forces between each other make the separation of various components quite difficult.
Using general gel filtration chromatography or ion exchange chromatography and other separation methods, the various components cannot be effectively separated: only one broad peak is displayed on the ordinary gel filtration chromatography chromatogram

Method used

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  • Method of separating protein aggregate in mixed liquid of denatured protein/incusion protein renaturation
  • Method of separating protein aggregate in mixed liquid of denatured protein/incusion protein renaturation
  • Method of separating protein aggregate in mixed liquid of denatured protein/incusion protein renaturation

Examples

Experimental program
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Effect test

Embodiment 1

[0018] Embodiment 1: Separating the protein aggregates in the denatured natural chicken egg white lysozyme protein refolding mixture to obtain a single-component natural chicken egg white lysozyme protein, the steps are as follows:

[0019] 1. Collect the natural chicken egg white lysozyme protein mixture after refolding, which contains correctly folded and refolded proteins, unfolded or misfolded proteins, and protein aggregates;

[0020] 2. Separation of protein aggregates in the natural chicken egg white lysozyme protein mixture after refolding

[0021] 1) Add 0.15 mol / L NaCl and 2 mol / L urea to 0.1 mol / L Tris-HCl, pH 8.7 gel filtration chromatography buffer to prepare a solution containing 0.15 mol / L NaCl and 2 mol / L The gel filtration chromatography buffer of urea of ​​L: and equilibrate Superdex 75 (10 / 30) gel filtration chromatography column with this buffer;

[0022] 2) Put the collected natural chicken egg white lysozyme protein mixture into the column, and use the a...

Embodiment 2

[0024] Example 2: The natural chicken egg white lysozyme protein after refolding was eluted with a buffer containing denaturant and salt ions, and the formation of refolded protein components was studied using a buffer without denaturing agents and salt ions as a control :

[0025] The gel filtration chromatography column used is a Superdex 75 prepacked column, and the elution flow rate is 0.4mL / min. figure 2 Curve 1 in the figure is: when the natural chicken egg white lysozyme protein after refolding was eluted using the gel filtration chromatography buffer solution without denaturing agent and salt ion, it was found that there was only one elution peak (the peak after the ion was peak), and the peak time is relatively late, suggesting that adsorption has occurred between the components of the three peaks and with the chromatographic column;

[0026]When the gel filtration chromatography buffer used contains a low concentration of NaCl, when the concentration of urea in the...

Embodiment 3

[0029] Example 3, using Tris-HCl buffer solution containing 1.5mol / L guanidine hydrochloride and 0.1mol / L potassium carbonate (or 0.1mol / L potassium acetate) to elute the protein in the mixed solution after human lysozyme inclusion body refolding Aggregate, make the natural chicken egg white lysozyme protein of single component, can obtain the similar separation effect with embodiment 1.

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Abstract

The method for separating protein aggregates in the denatured protein / inclusion body protein refolding mixture involved in the present invention is characterized in that, during the protein refolding process, the denatured refolded protein solution contains proteins in different states, and will contain proteins in different states The refolding protein solution of the protein is separated by gel filtration chromatography using gel filtration chromatography technology, and 1 to 3 mol of denaturant and 0.1 to 0.5 mol The salt ion; The denaturant is urea, guanidine hydrochloride, Triton or SDS; The salt ion comes from the + 、K + or NH 4 + Ionic hydrochloride, acetate, carbonate, phosphate or sulfate; it is easy to operate and can effectively separate various forms of components in the refolded protein and improve the purity of a single refolded protein.

Description

field of invention [0001] The invention relates to the field of biotechnology, in particular to a method for separating protein aggregates in denatured protein / inclusion body protein refolding mixture. Background technique [0002] No matter in protein laboratory research or industrial production, protein renaturation technology plays an important role. People currently express more than 4,000 kinds of proteins, among which the proteins expressed by Escherichia coli account for more than 90%, and most of these proteins are expressed in the form of inactive inclusion bodies, which must be dissolved with high-concentration denaturants. , to refold the target protein. Especially for the production of proteins with medicinal value, the renaturation efficiency of proteins expressed in inclusion bodies determines the cost and application of medicinal protein products. [0003] In the existing protein renaturation methods, the traditional dilution ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/34
Inventor 苏志国李明
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI