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

High-expression water-soluble heparinase I fusion protein and coding gene thereof

A technology of fusion protein and coding gene, applied in the field of genetic engineering, can solve problems such as poor water solubility, low activity, and poor water solubility of recombinant enzymes

Active Publication Date: 2014-08-20
SHANDONG UNIV
View PDF2 Cites 6 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when the pET expression system is used to express heparanase in Escherichia coli, the recombinant enzyme has always been poor in water solubility, easy to form inclusion bodies, requires complicated protein refolding treatment, and the refolded protein is unstable and easy to regenerate during storage. Problems such as precipitation time
In recent years, heparanase I has been widely cloned into different expression vectors and hosts, but still faces problems such as low expression, low activity, and poor water solubility. Gao's heparanase recombinant expression technology has important theoretical and practical significance

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
  • High-expression water-soluble heparinase I fusion protein and coding gene thereof
  • High-expression water-soluble heparinase I fusion protein and coding gene thereof
  • High-expression water-soluble heparinase I fusion protein and coding gene thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1, expression of heparanase I fusion protein pColdTF-HepI

[0027] 1. Cloning of Flavobacterium heparinase I coding sequence with signal peptide removed

[0028] The construction process of the expression vector pColdTF-HepI is as follows: figure 1 As shown, the specific process is as follows:

[0029] 1. Design and synthesis of primers

[0030] The DNA sequence of heparinase I from Flavobacterium heparinum (Su, H., Blain, F., Musil, R.A., Zimmermann, J.J., Gu, K. and Bennett, D.C. coli of hepB and hepC, genes coding for the glycosaminoglycan-degrading enzymes heparinase II and heparinase III, respectively, from Flavobacterium heparinum.Appl.Environ.Microbiol.1996, 62, 2723-2734), and then according to the removal of heparin encoding signal peptide base The DNA sequence design primer of Flavobacterium heparanase I, and introduce the recognition site of restriction endonuclease xba I and Nde I in primer sequence, used upstream and downstream primers are res...

Embodiment 2

[0056] Example 2, Purification of heparanase I fusion protein pColdTF-HepI by nickel column

[0057] pColdTF-HepI was transformed into Escherichia coli strain BL21(DE3) (purchased from Novagen, USA), and then induced expression with recombinase according to the operation steps provided by the company. And the HepI fusion protein was purified with Ni Sepharose6Fast Flow (GE) gel, and the purification conditions were operated according to the product manual of GE Company. The purification of recombinant pColdTF-HepI was detected by polyacrylamide gel electrophoresis, and the results were as follows: Figure 5 As shown in the No. 5 swimming lane of , the purified recombinant HepI fusion protein presents a single band on the electrophoresis gel, and the position coincides with the predicted molecular weight.

Embodiment 3

[0058] Embodiment 3, the enzyme activity assay of HepI fusion protein

[0059] The mass concentration was 1% heparin, pColdTF-HepI enzyme solution, 5 times buffer solution (250mM Tris-HCl, 500mM NaCl, 10mM CaCl 2, pH7.9) and water were mixed in the ratio of 2:1:2:5 (volume ratio), reacted at the optimum temperature and pH for 2-10min, and measured the enzyme activity by the aforementioned ultraviolet method (Yamagata, Saito et al.1968), while using the protein quantification kit purchased from Kangwei Century Company to measure the protein content of the HepI fusion protease solution, the results showed that the specific activity of the recombinant HepI fusion protein to heparin was 200U / mg.

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

PropertyMeasurementUnit
Extinction coefficientaaaaaaaaaa
Login to View More

Abstract

The invention relates to a high-expression water-soluble heparinase I fusion protein and a coding gene thereof. The amino acid sequence of the heparinase I fusion protein is as shown in SEQ ID NO. 2; the nucleotide sequence of the coding gene of the heparinase I fusion protein is as shown in SEQ ID NO. 1. According to the invention, a pColdTF vector is utilized to transform an expression gene of heparinase I, a section of nucleotide sequence expressing a pColdTF protein is added and the heparinase I fusion protein is obtained; the enzyme activity of the heparinase I fusion protein can reach 64000U / L of fermentation solution, the expression level can reach 320mg / L of fermentation solution and the specific enzyme activity can reach 200U / mg. Furthermore, the enzyme can further realize one-step purification of the fusion protein by nickel column separation.

Description

technical field [0001] The invention relates to a highly expressed water-soluble heparanase I fusion protein and its coding gene, belonging to the technical field of genetic engineering. Background technique [0002] Heparin / Heparan Sulfate (Hep / HS) both have the same main chain structure, which is composed of D-glucuronic acid / L-iduronic acid and N-acetylglucuronic acid through 20-100 Sugar amines are composed of straight chain polysaccharides connected by disaccharides. The hydroxyl group (-OH) in different parts of the sugar chain and the acetylation or sulfation of the 2-position amino group of N-acetylglucosamine make the structure of Hep / HS extremely complicated. (Castelli et al. 2004; Casu 2005). Hep / HS is widely distributed on the cell surface and extracellular matrix of mammals, and plays an important role in various life processes, such as: regulating blood vessel wall function, coagulation, inflammatory response and cell differentiation. [0003] Heparinase is a...

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): C12N9/88C12N15/62C12N15/74C12N15/81C12N15/75C12N15/80C12N15/82C12N15/85C12N1/21C12N1/15C12N1/19
CPCC12N9/88C12Y402/02007
Inventor 李福川赵梅韩文君王文爽彭立正
Owner SHANDONG UNIV
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