Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Renaturation and purification method of recombinant human granulocyte colony stimulating factor

A colony-stimulating factor and purification method technology, which is applied in the downstream protein purification field of biomedicine and bioengineering, can solve the problems of complex operation, low renaturation rate, and long time

Inactive Publication Date: 2019-07-23
JIANGSU AOSAIKANG PHARMA CO LTD
View PDF7 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the implementation of this method requires replacement steps, complicated operation and long time, and the concentration of inclusion protein bodies is low during renaturation, and the renaturation rate is low.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Renaturation and purification method of recombinant human granulocyte colony stimulating factor
  • Renaturation and purification method of recombinant human granulocyte colony stimulating factor
  • Renaturation and purification method of recombinant human granulocyte colony stimulating factor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1: Construction of pET-9a-G-CSF prokaryotic expression strain

[0069] The human G-CSF gene sequence is shown in SEQ ID No.1. Synthesize the human G-CSF gene whose sequence is shown in SEQ ID No.1 according to conventional molecular cloning methods and introduce the terminator TAA and the NdeI and BamHI restriction sites at both ends of the target gene; use NdeI and BamHI to prokaryotic expression plasmid vector pET-9a and the human G-CSF gene fragment were double-digested respectively, and the double-digested pET-9a and the human G-CSF gene fragment were connected to obtain the pET-9a-G-CSF recombinant plasmid (such as figure 1 ) into the E.coli DH5α strain.

[0070] Inoculate the E.coli DH5α strain containing the pET-9a-G-CSF recombinant plasmid into LB medium containing 50 μg / mL kanamycin at an inoculum size of 1%, and cultivate overnight at 37°C on a shaker (180rpm) , using a plasmid-free endotoxin extraction kit (purchased from Biomega) to extract and pur...

Embodiment 2

[0072] 1. Preparation of rhG-CSF refolding solution

[0073] The strain used in the process is the Escherichia coli DH5α strain transformed with the pET9a / G-CSF plasmid in Example 1. After fermentation, the bacterial cells are collected, the cells are crushed by a high-pressure homogenizer, and the crude inclusion bodies are obtained by centrifugation, and then the crude inclusion bodies are washed and centrifuged to obtain preliminary purified inclusion bodies.

[0074] Take 60 g of initially purified inclusion bodies, denature and dissolve them with inclusion body lysate (6 mol / L guanidine hydrochloride, 0.15 mol / L Tris, pH 8.0) at a solid-to-liquid ratio of 1:10 (g / mL), and stir at room temperature Add DTT (dithiothreitol) to a final concentration of 7 mmol / L until it becomes clear and transparent, continue stirring for more than 30 minutes, and filter through a 0.22 μm filter to obtain a solution of inclusion bodies.

[0075] Slowly pump the inclusion body solution into 1...

Embodiment 3

[0095] 1. Preparation of rhG-CSF refolding solution

[0096] The bacterial strain used in the process is Escherichia coli DH5α strain transformed with pET9a / G-CSF plasmid. After fermentation, the bacterial cells are collected, the cells are crushed by a high-pressure homogenizer, and the crude inclusion bodies are obtained by centrifugation, and then the crude inclusion bodies are washed and centrifuged to obtain preliminary purified inclusion bodies.

[0097] Take 60 g of initially purified inclusion bodies, denature and dissolve them with inclusion body lysate (5 mol / L guanidine hydrochloride, 0.1 mol / L Tris, pH 9.0) at a solid-to-liquid ratio of 1:10 (g / mL), and stir at room temperature Add DTT (dithiothreitol) to a final concentration of 10mmol / L until it becomes clear and transparent, and continue to stir for more than 30 minutes. After filtering through a 0.22μm filter, the inclusion body solution is obtained.

[0098] Slowly pump the inclusion body solution into 10L of...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides a renaturation and purification method of a recombinant human granulocyte colony stimulating factor. The renaturation and purification method comprises the steps of (1) preparing inclusion body dissolving liquid; (2) adding the inclusion body dissolving liquid to renaturation buffer liquid, adding cystine and cysteine, and performing renaturation; and (3) applying the obtained renaturation liquid to a weak cation chromatography column with a composite ligand for purification. The purification technology is simple and convenient to operate, short in consumption time, andlow in equipment requirement; the cost is saved, and the purification technology is suitable for amplified production scale; and besides, during renaturation, inclusion protein bodies are high in concentration and high in renaturation rate, and after chromatography purification, protein purity is high.

Description

technical field [0001] The invention relates to the field of downstream protein purification of biomedicine and bioengineering, in particular to a method for renaturation and purification of recombinant human granulocyte colony stimulating factor (rhG-CSF). Background technique [0002] Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic growth factor that regulates the maturation and growth of granulocytes (especially neutrophils) in mammals. It promotes the release of mature neutrophils into the peripheral blood by stimulating the differentiation and maturation of granulocyte-macrophage colony-forming units (CFU-GM) to granulocyte-colony-forming units (CFU-G), thereby promoting neutrophil growth. It is clinically used to treat neutropenia caused by various reasons, which has great economic and social significance. [0003] In 1985, Welte.K successfully purified and refined human granulocyte colony-stimulating factor (hG-CSF) from the culture supernata...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C07K14/535C07K1/14C07K1/18
CPCC07K14/535
Inventor 赵俊孟凯特张道平张林张瑜王晓慧李昕孙中涛方雅文勇
Owner JIANGSU AOSAIKANG PHARMA CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com