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Method for promoting protein renaturation by increasing charge density of microspherical medium through ligand modification

A charge density and protein technology, applied in the field of protein renaturation, can solve the problems of limited charge density and poor mechanical strength, and achieve the effects of increased charge density, easy removal, and high mechanical strength

Inactive Publication Date: 2014-02-26
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the agarose gel medium has a large specific surface area and can be modified into a high charge density medium, but its mechanical strength is poor; compared with the agarose gel medium, the non-porous monodisperse PGMA-PEIL microsphere medium has better Mechanical strength, and can be modified into superparamagnetic microspheres to greatly simplify the separation operation, but limited by the small specific surface area of ​​the non-porous microsphere medium and the modification method itself, the charge density of the PGMA-PEIL microsphere medium is limited, limiting Auxiliary refolding effect

Method used

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  • Method for promoting protein renaturation by increasing charge density of microspherical medium through ligand modification
  • Method for promoting protein renaturation by increasing charge density of microspherical medium through ligand modification
  • Method for promoting protein renaturation by increasing charge density of microspherical medium through ligand modification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Charge density of ligand remodified microsphere medium PGMA-PEIL-DEAE under different modification conditions

[0055]Synthesize the PGMA microsphere medium and modify polyethyleneimine PEIL with an average molecular weight of 60,000 on its surface to prepare a primary modified microsphere medium PGMA-PEIL with a charge density of 287 ± 9 μmol / g. See the literature (Mono-sized microspheres modified with poly(ethyleneimine)facilitate the refolding of like-charged lysozyme. Reactive and Functional Polymers, 2012, 72, 889-896).

[0056] The preparation method of the ligand remodified microsphere medium PGMA-PEIL-DEAE is as follows: Weigh three parts of the PGMA-PEIL microsphere medium with a mass of 1.0, 2.0 and 3.0 g, respectively, and transfer them into 50 mL Erlenmeyer flasks; After adding 5mL, 10mL and 15mL of DEAE solution with a concentration of 0.1mol / L, 0.5mol / L and 1.5mol / L respectively, then add 5mL, 10mL and 15mL of NaOH solution with a concentration ...

Embodiment 2

[0057] Example 2: Charge density of ligand remodified microsphere medium PGMA-PEIL-PEIS under different modification conditions

[0058] Synthesize the PGMA microsphere medium and modify polyethyleneimine PEIL with an average molecular weight of 60,000 on its surface to prepare a primary modified microsphere medium PGMA-PEIL with a charge density of 287 ± 9 μmol / g. See the literature (Mono-sized microspheres modified with poly(ethyleneimine)facilitate the refolding of like-charged lysozyme. Reactive and Functional Polymers, 2012, 72, 889-896).

[0059] The preparation method of the ligand-remodified microsphere medium PGMA-PEIL-PEIS is as follows: Weigh three parts of PGMA-PEIL microsphere medium with a mass of 1.0, 2.0 and 3.0 g, respectively, and transfer them into 50 mL Erlenmeyer flasks; Add 10mL, 20mL and 30mL of 50mmol / L boric acid / sodium borate buffer solution with a volume fraction of 10% glutaraldehyde and pH 9.0, and react at 25°C and 170rpm for 4h; Deionized water ...

Embodiment 3

[0060] Example 3: Ligand remodified microsphere medium PGMA-PEIL-DEAE assisted renaturation of 1 mg / mL reduced denatured lysozyme

[0061] Lysozyme is positively charged under the condition of pH 8.5 refolding, so the positively charged ligand remodified microsphere medium PGMA-PEIL-DEAE synthesized by the present invention can assist lysozyme refolding. In this example, the ligand remodified microsphere medium PGMA-PEIL-DEAE synthesized in Example 1 with a charge density of 824±28 μmol / g was selected to assist lysozyme renaturation, and under the same conditions without adding the microsphere medium and The primary modified microsphere medium PGMA-PEIL with a charge density of 287±9 μmol / g was added to assist lysozyme refolding as a control. Wherein, on the surface modification of PGMA microsphere medium, the average molecular weight is 60000 polyethyleneimine PEIL preparation charge density is the method for the primary modified microsphere medium PGMA-PEIL of 287 ± 9 μ mol / ...

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Abstract

The invention relates to a method for promoting protein renaturation by increasing the charge density of a microspherical medium through secondary ligand modification, belonging to a protein renaturation technology in the technical field of biology. A ligand of the microspherical medium is secondarily modified on the basis of primary modification of the microspherical medium, so that the charge density of the microspherical medium with epoxy groups on the surface is increased. The electrostatic repulsion action between the microspherical medium and a protein molecule with the same charge number is further enhanced through modifying the microspherical medium again by using the ligand with higher charge density, so that the aggregation of proteins is effectively inhibited, and the renaturation yield is greatly increased. Compared with a primarily-modified microspherical medium assisted protein renaturation method, the method not only has the characteristics of low medium consumption and high protein renaturation yield, but also has better tolerance for environments such as high salinity and the like.

Description

technical field [0001] The invention relates to a technology for ligand remodification to increase the charge density of a microsphere medium to promote protein renaturation, and belongs to the protein renaturation technology in the field of biotechnology. Background technique [0002] The rapid development of genetic engineering technology has provided important technical support for humans to decipher major diseases and develop new drugs. At present, the development of genetically engineered drugs has gradually entered a mature stage, and the production of such drugs must rely on suitable recombinant gene expression systems. Bacterial expression systems are widely used because of their advantages of high expression and easy amplification. However, when using this expression system to produce recombinant proteins, misfolding and aggregation often occur in cells due to excessive expression of the target protein, resulting in inclusion bodies. Inclusion bodies have no biolo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/14
Inventor 孙彦杨春燕史清洪董晓燕
Owner TIANJIN UNIV
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