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Method for separating polypeptide with tris ligand and agarose medium

A technology of tris and agarose, which is applied in the field of peptide separation and purification, can solve the problems of low reusability, poor biocompatibility, and low sample load of reversed-phase chromatography media, and achieve easy scale-up and recovery The effect of high efficiency and high sample load

Inactive Publication Date: 2010-01-20
北京康铭优盛生化技术有限公司
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  • Summary
  • 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 with tris ligand and agarose medium
  • Method for separating polypeptide with tris ligand and agarose medium
  • Method for separating polypeptide with tris ligand and agarose medium

Examples

Experimental program
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Embodiment 1

[0031]Example 1. Separation of three kinds of glycine oligopeptide mixtures using high cross-linking agarose gel hydrophilic adsorption chromatography medium with 12% concentration of Tris hydroxymethylaminomethane ligand

[0032] 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;

[0033] 2) Preparation of mobile phase: preparation of acetonitrile-water (20:80) solution, ultrasonic degassing for 30 minutes, and standing for 1 hour;

[0034] 3) The highly cross-linked agarose gel hydrophilic adsorption chromatographic medium with 12% concentration of trishydroxymethylaminomethane ligand is wet-packed in 20% ethanol preservation solution, the inner diameter of the chromatographic column is 10mm, and the column bed volume is 24mL. The pressure of the medium is 2.5MPa, and the volume of the initial mobile phase eq...

Embodiment 2

[0038] Example 2, Separation of lysozyme hydrolyzate with high cross-linking degree agarose gel hydrophilic adsorption chromatographic medium with 12% concentration of trishydroxymethylaminomethane ligand

[0039] 1) Preparation of lysozyme (Lysozyme) trypsin hydrolyzate: 5mg BPP dissolved in 1mL 20mM NH 4 HCO 3 Buffer (pH8), add 250μl 10mM dithiothreitol (DTT), incubate at 50°C for 30 minutes, cool to room temperature, add 250μl 30mM iodoacetamide, incubate for 60 minutes in the dark, follow the enzyme and protein ratio of 1:50 Add 500 μl of trypsin (without chymotrypsin activity) solution, mix well, incubate overnight at 37°C for about 15 hours, add 250 μl of acetonitrile to stop digestion, centrifuge the reaction solution at 1500 rpm for 10 minutes at 4°C, and filter the supernatant through 45 μm Membrane filtration standby;

[0040] 2) Preparation of mobile phase: preparation of acetonitrile-water (30:70) solution, ultrasonic degassing for 30 minutes, and standing for 1 ...

Embodiment 3

[0046] 8) According to the results of mass spectrometry, the fragment corresponding to the elution peak No. 2 is GTDVQAWIR, the fragment corresponding to the elution peak No. 4 is IVSDGNGMNAWVAWR, the fragment corresponding to the elution peak No. 5 is NTDGSTDYGILQINSR, and the fragment corresponding to the elution peak No. 8 is NLCNIPCSALLSSDITASVNCAK,而按照溶菌酶的氨基酸序列(KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFNTQATNRNTDGSTYGILQINSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDGNGMNAWVAWRNRCKGTDVQAWIRGCRL),推断由胰酶水解应产生18个片段,一些单个氨基酸如Arg和Lys可能由于吸附太强而未被洗脱,其它小片段在这一流动相 If they are not separated under certain conditions, the ratio of the mobile phase can be further adjusted to achieve the separation of the remaining fragments. Example 3. Separation and purification of solid-phase synthesis of oxytocin mixture using high cross-linking degree agarose gel hydrophilic adsorption chromatography medium with 12% concentration of trishydroxymethylaminomethane ligand

[0047] 1) Solid phase synthesis of oxyt...

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Abstract

The invention relates to a method for separating and purifying polypeptide with tris ligand and high-concentration high-crosslinking degree agarose gel hydrophilic adsorption chromatography medium. By adopting the tris ligand and the medium which takes the high-concentration high-crosslinking degree agarose gel as base material, and taking a hydrophilic adsorption chromatography theory which takes hydrogen bond interaction as a characteristic, the method further separates and purifies high-purity polypeptide from biological tissue extractive and lactalbumin hydrolysate or polypeptide synthetical mixture, thereby having the advantages of high selectivity, simple purifying technology, high sample carrying capacity, high medium repeat utilization ratio, etc. The method overcomes the defects of complex steps, the low repeat utilization ratio of the reversed phase chromatography medium, hard scalization, low sample carrying capacity, bad biocompatibility, etc. The purity of the obtained polypeptide fraction is ensured with a reversed phase high-efficient liquid phase chromatography, and the sequence and the molecular weight are ensured by mass spectrum.

Description

technical field [0001] The invention relates to a method for separating and purifying polypeptides, in particular to a method for separating and purifying polypeptides by agarose gel hydrophilic adsorption chromatography with trishydroxymethylaminomethane ligand, high concentration and high cross-linking degree. 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...

Claims

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Application Information

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IPC IPC(8): C07K1/22
Inventor 孟桂凤邢思亮
Owner 北京康铭优盛生化技术有限公司
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