Purification and renaturation method of Notch ligand Delta-like1 fusion protein

A fusion protein and ligand technology, applied in the field of denatured protein renaturation, can solve the problems of cumbersome process, high cost of stationary phase, unsuitable for mass production, etc., and achieve the effect of overcoming high cost, short purification process cycle and low cost

Inactive Publication Date: 2015-04-01
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although, compared with ion-exchange chromatography, high-specificity affinity chromatography can bring higher purity multiples to the purification and refolding of recombinant proteins, but, in addition to the disadvantage of high cost of stationary phase, the recombinant protein During the construction of the vector, purification tags such as GST and His need to be added, and after renaturation is completed, the protein and purification tags must be separated by RPLC [Wilkinson RJ, et al, Protein Expres Purifi.2004; 35: 334-343], the process Cumbersome and not suitable for mass production

Method used

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  • Purification and renaturation method of Notch ligand Delta-like1 fusion protein
  • Purification and renaturation method of Notch ligand Delta-like1 fusion protein
  • Purification and renaturation method of Notch ligand Delta-like1 fusion protein

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Embodiment 1

[0030] A method for purification and renaturation of Notch ligand Delta-like1 fusion protein, comprising the following steps:

[0031] 1) The inclusion bodies containing HD1R were washed with washing buffer several times, centrifuged, and dissolved in strong denaturant buffer to obtain crudely separated HD1R protein;

[0032] 2) Directly inject the denatured extract containing human HD1R protein into a column equilibrated with mobile phase A (20mmol / LPBS, pH8.0), and then use mobile phase B containing 20mmol / LPBS, 1mol / LNaCl, pH7.5 Isocratic elution, collecting target peak chromatographic fractions to obtain renatured and purified HD1R chromatographic fractions;

[0033] 3) Directly inject the collected chromatographic fraction of the target peak, further desalt with a chromatographic column equilibrated with 20mmol / LPBS mobile phase, and continue elution for 30min at a flow rate of 1.5mL / min to obtain an active chromatographic fraction with a higher degree of renaturation and...

Embodiment 2

[0035] A method for purification and renaturation of Notch ligand Delta-like1 fusion protein, comprising the following steps:

[0036] 1) The inclusion bodies containing HD1R were washed with washing buffer several times, centrifuged, and dissolved in strong denaturant buffer to obtain crudely separated HD1R protein;

[0037] 2) Directly inject the denatured extract containing human HD1R into a chromatographic column equilibrated with mobile phase A (40mmol / LPBS, or 20-40mmol / LTris, pH7.6), and then use a column containing 40mmol / LTris, 1mol / LNaCl, pH8 .0 mobile phase B isocratic elution, collect the chromatographic fraction of the target peak, and obtain the chromatographic fraction of HD1R after renaturation and purification;

[0038] 3) Directly inject the collected chromatographic fraction of the target peak, further desalt with a chromatographic column equilibrated with 20mmol / LPBS mobile phase, and continue elution for 30min at a flow rate of 1.5mL / min to obtain an activ...

Embodiment 3

[0040] A method for purification and renaturation of Notch ligand Delta-like1 fusion protein, comprising the following steps:

[0041] 1) The inclusion bodies containing HD1R were washed with washing buffer several times, centrifuged, and dissolved in strong denaturant buffer to obtain crudely separated HD1R protein;

[0042] 2) Directly inject the denatured extract containing human HD1R protein into the chromatographic column equilibrated with mobile phase A (30mmol / LPBS, pH7.7), and then use the mobile phase B line containing 30mmol / LPBS, 1mol / LNaCl pH7.8 Isocratic elution, collecting target peak chromatographic fractions to obtain renatured and purified HD1R chromatographic fractions;

[0043] 3) Directly inject the collected chromatographic fraction of the target peak, use a chromatographic column equilibrated with 20mmol / LPBS mobile phase for further desalting, continue elution for 30min, flow rate 0.5mL / min, and obtain an active chromatographic fraction with a higher deg...

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Abstract

A purification and renaturation method of Notch ligand Delta-like1 fusion protein is characterized by comprising steps as follows: 1), an inclusion body containing HD1R is repeatedly washed and centrifuged by a washing buffer and is dissolved in a strong denaturant buffer to obtain crudely separated HD1R protein; 2), a denatured leaching solution containing human proinsulin is subjected to direct sample introduction to a flowing phase A balanced chromatographic column and is subjected to isocratic elution by a flowing phase B line of PBS (poly butylenes succinate), 1 mol / L of NaCl or Tris and 1 mol / L of NaCl, target peak chromatographic fraction is collected, and renatured and purified HD1R chromatographic fraction is obtained; and 3), the collected target peak chromatographic fraction is subjected to direct sample introduction, is further desalinated by the flowing phase A balanced chromatographic column of 20 mmol / L of PBS and is continuously eluted for 30 min, the elution flow velocity is 0.5-1.5 ml / min, and the renatured and purified chromatographic fraction with higher purity and higher activity is obtained. According to the method, the process is simple, large-scale production can be realized, and the obtained fraction with high yield and high purity can provide a reliable experimental material for intensive study of Notch signal pathways.

Description

field of invention [0001] The invention belongs to the technical field of denatured protein refolding in biotechnology, in particular to a method for purifying and refolding Notch ligand Delta-like1 fusion protein, using ion exchange chromatography and size exclusion chromatography to purify and refold Notch ligand Delta-like1. like1 fusion protein. technical background [0002] Notch is named after Morgan discovered in Drosophila in 1916 that some mutations can cause a gap (Notch) at the edge of the Drosophila wing. Notch signaling is a way to control cell fate through local cell-to-cell interaction, which is highly conserved in evolution and widely expressed in embryonic and adult individual tissues. The signaling pathway includes ligand DSL (Delta / Serrate / Lag-2, DSL), receptors, downstream molecules and regulatory molecules. At present, four kinds of Notch receptor proteins have been found in mammals, namely Notch1-4; These receptors / ligands are highly homologous in st...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K1/18C07K1/16
Inventor 张萍韩骅晏贤春杨子岩
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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