Preparation and application of high performance hydrophobic interaction chromatography packing material taking cholesterol as aglucon

A technology of hydrophobic interaction and chromatography packing, which is applied in the field of preparation and application of high-efficiency hydrophobic interaction chromatography packing with cholesterol as ligand, which can solve the problem that denatured protein is easy to penetrate directly, denatured protein is difficult to elute, and protein renaturation efficiency is low. and other problems, to achieve the effect of simple and mature preparation process, easy control and scale-up, and good biocompatibility

Inactive Publication Date: 2015-08-19
NORTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The filler composed of the former ligand is easy to cause proteins, especially denatured proteins, to be difficult to elute
In the process of denatured protein refolding, the latter ligand with weak hydrophobicity will cause the decrease of adsorption capacity and the disadvantages of easy direct penetration of denatured protein, so that the refolding efficiency of protein is very low.

Method used

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  • Preparation and application of high performance hydrophobic interaction chromatography packing material taking cholesterol as aglucon
  • Preparation and application of high performance hydrophobic interaction chromatography packing material taking cholesterol as aglucon
  • Preparation and application of high performance hydrophobic interaction chromatography packing material taking cholesterol as aglucon

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] (1) Preparation of High Efficiency Hydrophobic Interaction Chromatography Packing

[0040] Using high-purity spherical silica gel as a blank matrix, first carry out acid treatment, reflux the silica gel with 20% hydrochloric acid at 120°C for 3-4 hours, cool, wash repeatedly with distilled water until the solution is neutral, and vacuum dry overnight. Then the acid-treated silica gel is activated with γ-glycidyloxypropyltrimethoxysilane (TM-560), that is, in a 50mmol / L sodium acetate solution, an appropriate amount of TM-560 is slowly added dropwise at 90°C After the silica gel was reacted for 4 hours, it was washed with 50 mmol / L sodium acetate solution, methanol and water in sequence, and dried in vacuum. The reaction between cholesterol and epoxidized silica gel was carried out in anhydrous ether and dry 1,4-dioxane solution, and 2.0 mL of boron trifluoride ether was added as a catalyst, and the reaction was carried out at 5 ° C for 4 h, and then with Wash with ethe...

Embodiment 2

[0046] Similar to Example 1, the difference is that step (3) refolds denatured lysozyme and simultaneously purifies it with high-efficiency hydrophobic interaction chromatography filler, and its mobile phase A (equilibrium buffer) used is 3.0mol / L (NH 4 ) 2 SO 4 , 50.0mmol / L KH 2 PO 4 , 3.0mol / L Urea, pH7.0, mobile phase B (refolding buffer) is 50.0mmol / L KH 2 PO 4 , 3.0mol / L Urea, pH7.0 for refolding elution, the elution method is a linear gradient of 0-100% B for 30min, and prolonging 100%B for 10min, the flow rate is 1ml / min, and the chromatographic peaks are collected, see image 3 b.

Embodiment 3

[0048] Similar to Example 1, the difference is that step (3) refolds and simultaneously purifies recombinant human tyrosine kinase ligand (rhFL) with high-efficiency hydrophobic interaction chromatography filler, and the inclusion body is dissolved in 8.0mol / L Urea, 1mmol / L EDTA, 50.0mmol / L DDT, pH 8.0 buffer solution to obtain denatured extract of rhFL inclusion body. Use mobile phase A (equilibrium buffer) as 3.0mol / L (NH 4 ) 2 SO 4 , 50.0mmol / L KH 2 PO 4 , 3.0mol / L Urea, pH7.0 balance, 250μL denatured extract was adsorbed and sampled, using 0-100% B (50.0mmol / L KH 2 PO 4 , 3.0mol / L Urea, pH7.0) linear gradient elution 30min, prolong 100%B 10min, flow rate is 1ml / min, collect chromatographic fraction, see Figure 4 .

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Abstract

The invention provides methods for preparing and applying a high performance hydrophobic interaction chromatography (HPHIC) packing material with a special function. Amphiprotic functional molecule cholesterol on a cell membrane is taken as a stationary phase aglucon, and surface chemical modification is performed on silica matrix, thus obtaining the biomimetic HPHIC packing material. An HPHIC column filled with the packing material adopts salt-water as an elution system, and can be applied to natural protein separation and renaturation and simultaneous purification of denatured lysozyme, a recombined human Flt3 ligand (rhFL) inclusion body and a recombined human stem cell factor (rhSCF).

Description

field of invention [0001] The invention relates to a preparation method of a high-efficiency hydrophobic interaction chromatographic filler with cholesterol as a ligand and its use in protein refolding and purification, and belongs to the intersection field of high-performance liquid chromatography technology and bioengineering technology. Background technique [0002] Cholesterol is a major component of biological membranes. In HPLC, using cholesterol molecules as chromatographic ligands and using surface chemical modification to synthesize chromatographic fillers has aroused great interest of chromatographic workers and has made certain progress [Witkiewicz Z., Mazur J.LC GC, 1990, 8 (3 ):224-236; Pesek J.J., Matyska M.T. Interface Science, 1997, 5(2):103-117]. According to the existing literature reports, the chromatographic packing materials with cholesterol as the ligand mainly focus on the research of reversed-phase liquid chromatography (RPLC), and its high-efficienc...

Claims

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Application Information

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IPC IPC(8): B01J20/22B01J20/30B01J20/281B01J39/16B01J39/08C12N9/36C07K14/705C07K14/475C07K1/20
Inventor 王骊丽屈倩袁洁杨毅聪
Owner NORTHWEST UNIV
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