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

Method for purifying chondroitinase ABC

A chondroitinase, heparin technology, applied in biochemical equipment and methods, enzymes, enzymes and other directions, can solve difficulties and other problems, and achieve the effect of high recovery rate and simple recovery rate

Active Publication Date: 2017-09-19
ADVANTEK SERUM LAB
View PDF2 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This makes designing affinity chromatography for ChABC more difficult compared to chondroitinase AC, chondroitinase B, and chondroitinase C

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
  • Method for purifying chondroitinase ABC
  • Method for purifying chondroitinase ABC
  • Method for purifying chondroitinase ABC

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] In the present invention, the identification of molecules that have a similar structure to the substrates of ChABC but are inert to enzymatic degradation is the first critical part of the invention. To test for polysaccharides that can actually bind to the active site of ChABC, a relative enzyme inhibition assay was performed on selected polysaccharides:

[0050] 490 [mu]L of substrate solution containing 0.2% chondroitin sulfate C, 0.2% selected polysaccharide and 50 mM pH 8.0 Tris HCl buffer was incubated at 37[deg.] C. for at least 15 minutes. 10 μL of ChABC with an activity of about 100 U / L was incubated alone for at least 3 minutes at 37°C. After incubation, mix the two solutions gently. The mixture was then incubated at 37°C for 20 minutes for enzymatic degradation of chondroitin sulfate. After 20 min, the reaction was terminated by heat inactivating ChABC in a 100 °C water bath for 2 min. Blanks were performed separately in the same manner except that the mixt...

Embodiment 2

[0070] To test whether ChABC specifically interacts with immobilized heparin, Affi Columns of heparin gel. The column was pre-equilibrated with 25 mM Tris-acetate (pH 7.3) prior to loading samples onto the column. ChABC was dissolved in 25 mM Tris-acetate (pH 7.5) and loaded onto the column. With (1) 25mM Tris-acetate (pH9.2), (2) 100mM Tris-acetate (pH9.2), (3) 60mM Tris-acetate (pH8) and (4) 100mM Tris-acetate The column was washed with hydrochloric acid (pH 7.5). None of these buffers could elute ChABC from the column. Whereas ChABC could be eluted from the column when a linear gradient of 0-0.3M NaCl was applied. Chromatograms such as figure 1 shown.

Embodiment 3

[0072] Using the genome of Proteus vulgaris as a template, the gene encoding chondroitinase ABC (SEQ ID NO: 1) was cloned by PCR, and the product was connected to the pET3a plasmid by T4DNA ligase, and then the recombinant plasmid was introduced into the E. coli strain by electroporation BL21(DE3). The transformed E. coli were inoculated on LB agar plates containing 50 μg / ml ampicillin to select successfully transformed colonies. Select a single colony, then inoculate it into LB medium containing 50 μg / ml ampicillin at 37 °C for 18 hours, and add the culture to sterile LB medium containing 50 μg / ml ampicillin at a ratio of 1:100 . The medium was further incubated at 37°C to OD600=0.8. Addition of 1 mM IPTG induced chondroitinase production, followed by further incubation for 4 hours. Harvest by centrifugation and store at -80 °C.

[0073] 10 grams of recombinant E. coli cells were resuspended in 25 mM Tris HCl (pH 7.0) and lysed by sonication for 5 minutes. Cell debris wa...

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 discloses a method for purifying chondroitinase ABC. According to the method, an affinity column, on which heparin is fixed, is used for obtaining purified chondroitinase ABC from a substrate containing chondroitinase ABC by means of a chromatography. The method can be used for obtaining the high-purity chondroitinase ABC and has the advantages of being simple and high in recovery rate.

Description

technical field [0001] The present invention relates to a method for purifying chondroitinase ABC (ChABC), in particular to a method for purifying ChABC from a ChABC-containing matrix. Background technique [0002] Chondroitin sulfate is the most abundant glycosaminoglycan in biological systems, consisting of a variety of sulfated and non-sulfated carbohydrate derivatives including sulfated glucuronic acid, N-acetylgalactosamine, iduronic acid, etc. . This polymer is often linked to proteins to form chondroitin sulfate proteoglycan (CSPG), which plays many structural and functional roles in biological systems (e.g., extracellular matrix that enables cell-cell interactions, directs or inhibits neural development, synthesizes cartilage and become structural components). The biosynthesis and maintenance of these molecules in proper position is crucial for the normal function of the human being. In fact, many diseases are related to the deficiency, accumulation and dislocatio...

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
Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88
CPCC12N9/88C12Y402/02004
Inventor 黄炳镠钟玮康
Owner ADVANTEK SERUM LAB
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