Heparanase III fusion protein and coding gene and expression method thereof

A technology of heparinase and protein, applied in the fields of genetic engineering and fermentation engineering, can solve problems such as unsatisfactory results, high purification costs, unfavorable industrial applications, etc.

Active Publication Date: 2011-01-12
TSINGHUA UNIV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] However, the research on the heterologous recombinant expression of heparanase III is very limited. So far, only Godavarti and Su et al. have reported recombinant expression of heparanase III, but the results are not ideal
Godavarti results show that the activity of recombinant heparanase III is very low, and most of them exist in insoluble form (80-85% are inclusion bodies) (Godavarti R, Davis M, Cooney C, Langer R, Sasisekharan R.Heparinase III from Flavobacterium heparinum: Cloning and Recombinant Expression in Escherichia coli.Biochem.Biophys.Res.Commun.1996, 225:751-758); the activity of heparinase III expressed in Su's study was improved, but one-step purification was not possible , resulting in relatively high purification costs, which is not conducive to industrial applications (Su H, blain F, Musil RA, Zimmermann JJF, Gu K, Bennett DC. Isolation and Expression in Escherichia 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)

Method used

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  • Heparanase III fusion protein and coding gene and expression method thereof
  • Heparanase III fusion protein and coding gene and expression method thereof
  • Heparanase III fusion protein and coding gene and expression method thereof

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Embodiment 1

[0038] Embodiment 1, expression of heparanase III fusion protein MBP-HepC

[0039] 1. Cloning of Flavobacterium heparinase III coding sequence with signal peptide removed

[0040] The construction process of the expression vector pMAL-hepC is as follows: figure 1 As shown, the specific process is as follows:

[0041] 1. Design and synthesis of primers

[0042] The DNA sequence of heparinase III of 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 the coding signal peptide base Design primers for the DNA sequence of Flavobacterium heparinase III heparinase, and introduce the recognition sites of restriction endonucleases BamH I and Pst I in the primer sequences, and the upstream and downst...

Embodiment 2

[0063] Example 2. Purification of heparanase III fusion protein MBP-HepC by amylose column

[0064] The fusion partner (fusion partner) maltose binding protein MBP utilized in the present invention can achieve one-step separation with amylose affinity adsorption. The specific steps of affinity separation are as follows: 100 mL of cells whose final concentration was 0.24 mM IPTG induced expression for 25 hours were centrifuged at 10,000 rpm for 5 minutes; at the same time, cells without induced expression were set as control. Then proceed with the following two options:

[0065] Option 1: Wash twice with Column buffer (20mM Tris-HCl, 200mM NaCl, pH7.5), resuspend in 5mL Column buffer, and perform sonication (300W output power, 3 seconds each time and intermittent 198 times in 3 seconds).

[0066] Option 2: Osmotic pressure shock. Resuspend the bacteria in 100 mL osmotic shock buffer I (20-40% sucrose, 30 mM Tris-HCl, 1 mM EDTA) for 15 minutes, and stir. Centrifuge at 10,000...

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Abstract

The invention discloses a heparanase III fusion protein and a coding gene and an expression method thereof. The fusion protein of the invention (named as MBP-HepC) is a protein in a) or b): a) a protein consisting of amino acid sequences shown by the 1-1028th site of a sequence 2 in a sequence table; and b) a protein derived from a), provided with heparanase III activity and obtained by substituting and / or deleting and / or adding one or more amino acids in an amino acid sequence of the sequence 2 in the sequence table. The coding gene of the protein is also within the protection range of the invention. By introducing the gene into protein heparanase expressed in recombinant escherichia coli, the enzymatic activity can reach 1,776.3 IU per liter of fermentation liquid, the expression amount can reach 240 mg per liter of fermentation liquid, and the specific enzymatic activity can reach 7.39 IU / mg. The invention also can realize further purification of the fusion protein through affinity separation.

Description

technical field [0001] The invention relates to a heparanase III fusion protein, its encoding gene and its expression method in the fields of genetic engineering and fermentation engineering. Background technique [0002] Heparinase (heparinase) is a class of polysaccharide lyase that acts on heparin (heparin) or heparan sulfate (heparan sulfate), found in many kinds of microorganisms, including Corynebacterium sp. (Gao Ningguo et al., Heparinase production Screening and fermentation conditions of bacteria, Acta Microbiology 1999 Vol.39:64-67), Sphingobacterium sp. 816), Bacillus subtilis (Wang Zhongyan, Screening of Heparanase-producing Bacteria and Research on the Properties of Crude Enzyme, Journal of Sichuan University (Natural Science Edition) 2002 Vol.39: 777-779), Bacillus circulans Bacillus circulans (Yasutaka Tahara et al., Purification and characterization of heparinase that degrades both heparin and heparin sulfate from Bacillus circulans BioSci.Biotechnol.Bioche...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21C12N9/42
Inventor 邢新会李晔叶逢春蒋培霞张翀冯权
Owner TSINGHUA UNIV
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