Method for preparing heparinase

A technology of heparinase and plasmid, applied in the direction of microorganism-based methods, biochemical equipment and methods, lyase, etc., can solve the problems of high market price, poor stability of heparinase, and difficulty in purification

Inactive Publication Date: 2011-09-28
GUANGYUAN HAIPENG BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Heparinase has poor stability, is not easy to purify, and is expensive in the market
According to foreign reports, the titer of

Method used

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Examples

Experimental program
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Embodiment Construction

[0007] An embodiment of the present invention comprises the following steps: the existing plasmids Hep1-PET-15b, HepII-PET-19b, and HepIII-PET-19b in the laboratory all have ampicillin resistance, which are respectively transformed into Escherichia coli JM109, and passed Gene sequencing, its sequence is completely consistent with the sequence information registered on GeneBank. The genes of heparanase I, II, and III contain open reading frames of 1155, 2319, and 1980 bp respectively, encoding protein precursors with 384, 772, and 659 amino acid sequences, respectively. After the expression of the precursors, under the action of aminopeptidase , cut out 21, 25 and 24 amino acid signal peptide sequences from Ala21-Gln22, Ser25-Gln26, and Ala24-Gln25, respectively, to form mature heparanase I, II, and III. After the sequencing is correct, the plasmid is then transferred into the large intestine Bacillus BL21, induced by low temperature, its protein molecular weights are 42KD, 84K...

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Abstract

The invention discloses a method for preparing heparinase. The method comprises the following steps of: selecting any of the conventional plasmids with ampicillin resistance, namely Hep1-PET-15b, HepII-PET-19b, and HepIII-PET-19b, importing into Escherichia coli JM109, transferring to Escherichia coli BL21 after the sequencing is correct so as to obtain an engineering bacteria strain for expressing the heparinase, culturing the engineering bacteria, inducing and expressing the heparinase, and purifying by using a nickel nitrilotriacetic acid (NTA) agarose gel column. By the method, the high-level expression can be realized, and the purity obtained by the purification is over 95 percent.

Description

technical field [0001] The invention relates to a preparation method of heparanase. Background technique [0002] Heparinase (heparinase) is a kind of enzyme network that can specifically cleave the glycosidic bonds of the main chain of heparin and heparinoids. It was originally discovered and isolated from Flavobacterium heparinum. As the only enzyme that can degrade heparin and heparan-like glycosaminoglycans, heparanase is closely related to pathological processes such as angiogenesis, tumor metastasis, and inflammation. At present, three kinds of heparinases have been isolated from Flavobacterium heparinus, namely heparinase I, heparinase II and heparinase III, among which heparinase I is the main one, and the other two kinds are less in content. These three enzymes degrade heparin and heparan sulfate as substrates, and finally produce oligosaccharide fragments. It has important uses in preparing low molecular weight heparin, eliminating heparin anticoagulants in extra...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/70C12R1/19
Inventor 王香李海生
Owner GUANGYUAN HAIPENG BIOLOGICAL TECH
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