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Method for separating polypeptide through hydrogen binding adsorption chromatography of quercitin aglucon and agarose

A technology of hydrogen bond adsorption and chromatographic separation, applied in the preparation methods of peptides, chemical instruments and methods, peptides, etc., can solve the problems of low reusability of reversed-phase chromatography media, poor biocompatibility, and low sample load, etc. Achieve easy scale-up, high recovery, and high sample load

Inactive Publication Date: 2011-10-12
HANGZHOU HUAJIN PHARMA +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] The purpose of the present invention is to simplify the steps of polypeptide separation and overcome the disadvantages of traditional methods such as complex steps, low reusability of reversed-phase chromatography media, difficulty in scale-up, low sample load, and poor biocompatibility.

Method used

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  • Method for separating polypeptide through hydrogen binding adsorption chromatography of quercitin aglucon and agarose
  • Method for separating polypeptide through hydrogen binding adsorption chromatography of quercitin aglucon and agarose

Examples

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Embodiment 1

[0030] Example 1. Separation of three glycine oligopeptide mixtures using high cross-linking degree agarose gel hydrogen bond adsorption chromatography medium with 12% concentration of quercetin ligand

[0031]1) Preparation of glycine oligopeptide mixture: Weigh standard products 0.6mgGly-Gly, 1.2mgGly-Gly-Gly and 0.4mgGly-Gly-Gly-Gly-Gly, mix them and dissolve them in 5mL mobile phase, sonicate, 0.45μm Membrane filtration;

[0032] 2) Preparation of mobile phase: preparation of acetonitrile-water (10:90) solution, ultrasonic degassing for 30 minutes, and standing for 1 hour;

[0033] 3) Highly cross-linked agarose gel hydrogen bond adsorption chromatography medium with 12% quercetin ligand is wet-packed in 20% ethanol preservation solution, the inner diameter of the chromatography column is 10mm, the column bed volume is 24mL, and the medium pressure 2.5MPa, the volume of the initial mobile phase equilibrium chromatography column is 2 column beds;

[0034] 4) Inject 500 μL...

Embodiment 2

[0037] Example 2. Using quercetin ligand with 12% concentration of high cross-linking degree agarose gel hydrogen bond adsorption chromatography medium to separate and purify liquid phase synthesis of thymopentin mixture

[0038] 1) liquid phase synthesis of thymopentin mixture;

[0039] 2) Preparation of mobile phase: preparation of acetonitrile-water (30:70) solution, ultrasonic degassing for 30 minutes, and standing for 1 hour;

[0040] 3) Highly cross-linked agarose gel hydrogen bond adsorption chromatographic medium with 12% concentration of quercetin ligand, agarose gel medium wet-packed column, the inner diameter of the chromatographic column is 10mm, the column bed volume is 24ml, and the pressure resistance is 2.5MPa. The chromatographic column equilibrates 2 column bed volumes with the initial mobile phase;

[0041] 4) Inject 1ml of the liquid-phase synthetic thymopentin mixture sample through the automatic sampling valve;

[0042] 5) Isocratic elution, generally w...

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Abstract

The invention relates to a method for separating and purifying polypeptide through a hydrogen binding adsorption chromatographic medium of quercitin aglucon and high-concentration and high-crosslinking degree agarose gel. The quercitin aglucon and the high-concentration and high-crosslinking degree agarose gel serve as a medium of a matrix; and the high-purity polypeptide is further separated and purified from a biological tissue extract, a protein hydrolysate or a polypeptide synthesis mixture based on the principle of the hydrogen binding adsorption chromatography characterized by hydrogen binding mutual effect. The method has the characteristics of high selectivity, simple purification process, large sample carrying amount, high medium recycling degree and the like. The defects that the traditional method has complex steps, low reversed-phase chromatographic medium recycling degree, difficult mass production, low sample carrying amount, poor biocompatibility and the like are overcome. The purity of the obtained polypeptide fractions is determined through reversed-phase high efficiency liquid chromatography, and the sequence and the molecular weight of the polypeptide are determined through a mass spectrum.

Description

technical field [0001] The invention relates to a polypeptide separation and purification method, in particular to a quercetin ligand, high-concentration, high-crosslinking degree agarose gel hydrogen bond adsorption chromatography separation and purification method for polypeptides. Background technique [0002] There are a large number and variety of biologically active polypeptides in nature, which play a very important role in regulating various life activities, involving various toxins, hormones, antimicrobial peptides, pheromones, cytokines, etc., and are widely involved in molecular recognition, signal transduction, etc. Guidance, enzyme activity regulation, immune regulation, cell differentiation and individual development regulation and other processes. The development and utilization of biologically active peptides has been paid more and more attention by industries such as medicine, food, and cosmetics. Especially in the pharmaceutical industry, a large number of ...

Claims

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Application Information

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IPC IPC(8): C07K1/22
Inventor 顾铭高留根谷海涛周天琼周正兵俞保彬
Owner HANGZHOU HUAJIN PHARMA
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