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A highly expressed water-soluble heparanase I fusion protein and its coding gene

A fusion protein and coding gene technology, applied in the field of genetic engineering, can solve the problems of low expression, poor water solubility, low activity, etc.

Active Publication Date: 2016-05-04
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, when the pET expression system is used to express heparanase in Escherichia coli, the recombinant enzyme has always had poor water solubility, easy to form inclusion bodies, requires complex protein renaturation treatment, and the renatured 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

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  • A highly expressed water-soluble heparanase I fusion protein and its coding gene
  • A highly expressed water-soluble heparanase I fusion protein and its coding gene
  • A highly expressed water-soluble heparanase I fusion protein and its coding gene

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Experimental program
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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. Isolation and expression in Escherichia aliofhep Bandhep C, genescoding for theglycosaminoglycan-degrading enzyme II, genecoding for theglycosaminoglycan-degrading enzyme II, genecoding for theglycosaminoglycan-degrading enzyme II, heparinase I was obtained through Genbank query , fromFlavobacterium heparinum.Appl.Environ.Microbiol.1996, 62, 2723-2734), and then design primers according to the DNA sequence of Flavobacterium heparinase I that encodes the base of the signal peptide...

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 NiSepharose6FastFlow (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, 500mMNaCl, 10mM CaCl 2 , pH7.9) and water were mixed according to the ratio of 2:1:2:5 (volume ratio), reacted at optimum temperature and optimum pH for 2-10min, and measured enzyme activity according to the aforementioned ultraviolet method (Yamagata, Saito et al. .1968), while measuring the protein content of the HepI fusion protease liquid with the protein quantitative kit purchased from Kangwei Century Company, the results showed that the specific activity of the recombinant HepI fusion protein to heparin was 200U / mg.

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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-acetylglucose through 20-100 Amines are composed of straight-chain polysaccharides connected by disaccharides. The hydroxyl (-OH) in different positions in 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 an important tool...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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
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