An efficient protein polymerization method

A protein and high-efficiency technology, applied in the field of biomedicine, can solve the problems of cumbersome removal process and long time, and achieve the effects of short polymerization reaction time, reduced ratio and high polymerization efficiency

Inactive Publication Date: 2018-04-10
SHANXI LIFEGEN
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The removal process is cumbersome and takes a long time

Method used

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  • An efficient protein polymerization method
  • An efficient protein polymerization method

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Experimental program
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Embodiment 1

[0045] Embodiment 1 prepares polyprotein with anion exchange resin

[0046] (1) Pretreatment of the medium: Take 10ml of DEAE agarose (anion exchange resin) and add 20ml of 0.02M glutaraldehyde to react with possible groups on the solid phase medium that can react with aldehyde groups for 1 hour. Add 20ml of 0.8M sodium borohydride to reduce the resulting product and excess glutaraldehyde, centrifuge at 1000r / min and discard the supernatant, and repeat washing with buffer 5 times. The unreacted polyaldehydes were terminated, polyaldehydes and reducing agents in the system were washed away with 15 mM phosphate buffer, and the pH of the solid phase medium was adjusted to 9.

[0047] (2) Pretreatment of polyaldehyde-based molecules: glutaraldehyde and glycerol are mixed according to the molar ratio of 1:40, the volume after mixing is 15ml, and 10 microliters of 1.5M hydrochloric acid are added for catalysis, so that the pH of the solution is 6. The reaction temperature is 40°C, ...

Embodiment 2

[0052] Embodiment 2 prepares protein with cation exchange resin

[0053] (1) Pretreatment of the medium: Take 10ml of H-type cation exchange resin and add 20ml of 0.05M malondialdehyde to react with possible groups on the solid phase medium that can react with aldehyde groups. After reacting for 0.5 hours, add 20ml of 0.5M dimethylaminoborane, reduce the resulting product and excess malondialdehyde, centrifuge at 1000r / min and discard the supernatant, and repeat washing with 15mM HEPES buffer 5 times. And stop the unreacted polyaldehyde, wash away other residues in the system with buffer, and adjust the pH of the solid phase medium to 6.

[0054] (2) Pretreatment of polyaldehyde-based molecules: MDA and ethylene glycol are mixed according to the molar ratio of 1:60, the final volume is 15ml, and 10 microliters of 1.5M sulfuric acid are added for catalysis, so that the pH of the solution is 5.5 , the reaction temperature was 4° C., and the reaction time was 80 min. .

[0055...

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Abstract

The invention proposes a method for efficiently polymerizing proteins, which can greatly reduce the proportion of unpolymerized proteins in a mixture of polymerized proteins, without the need for subsequent steps of removing unpolymerized proteins, and has short polymerization reaction time and simple polymerization process. The method comprises the following steps: firstly mixing a solid-phase adsorption medium with protein, and the solid-phase adsorption medium is anion exchange resin or cation exchange resin; after the protein is adsorbed on the solid-phase adsorption medium, washing to remove Adsorb the protein on the medium; then add polyaldehyde molecules to polymerize the protein; after the polymerization is completed, add a reducing agent to reduce the double bond of Schiff's base to a stable single bond, wash to remove other reactants, and obtain a polymerized protein.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a method for rapidly polymerizing proteins. Background technique [0002] Many natural proteins are not easily absorbed by the body due to their small molecular weight. After increasing their molecular weight or molecular radius through polymerization or modification, they can also form new properties in addition to retaining some of their original biological properties, such as colloid osmotic pressure. Half-life increase, etc., can be applied to the field of biomedicine. [0003] At present, protein polymerization to increase its molecular weight is mainly used in the field of hemoglobin oxygen carriers. The amino groups on the surface of the protein are cross-linked by Schiff's base reaction through polyaldehyde molecules to increase the molecular weight of the protein. The polymerization method is usually to directly add polyaldehyde-based molecules such as glutaraldehyde and oxidize...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/00
CPCC07K1/00
Inventor 严坤平冯坤陈超
Owner SHANXI LIFEGEN
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