Heparinase-producing Pseudomonas stutzeri strain and heparinase derived therefrom
AssigneeShenzhen Hepalink Pharmaceutical Group Co., Ltd.
InventorsLi Li, Jiake Bai, Jinliang Gao, Xiaolai Ma
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Abstract
The invention relates to the field of bioengineering. In particular, the invention relates to a heparinase-producing Pseudomonas stutzeri strain and a heparinase derived therefrom. Furthermore, the invention relates to the preparation and use of the heparinase.
Claims18
A recombinant heparinase comprising an amino acid sequence shown in SEQ ID NO:2,
- wherein the recombinant heparinase comprises a linker or is fused to a tag, wherein the linker and/or tag facilitates the production of the recombinant heparinase in a host cell of interest, increases the amount of expression or increases the soluble expression, facilitates the isolation and purification of the recombinant heparinase, but do not substantially affect the activity of the recombinant heparinase.
An expression construct, comprising a nucleotide sequence encoding the heparinase comprising an amino acid sequence shown in SEQ ID NO:2, operably linked to an expression control sequence.
A method for producing heparinase which comprises:
- culturing a Pseudomonas stutzeri strain deposited at the China Center for Type Culture Collection under the Accession Number CCTCC M 2017174;
- lysing the cultured cells of the Pseudomonas stutzeri strain;
- and isolating and/or purifying the heparinase from the lysate.
A method for producing heparinase, wherein the method comprises the steps of:
- a) culturing a Pseudomonas stutzeri strain deposited at the China Center for Type Culture Collection under the Accession Number CCTCC M 2017174 and collecting the cultured bacterial cells;
- b) resuspending and lysing the cells obtained in step a) in a buffer, and collecting the supernatant after centrifugation;
- c) precipitating the supernatant obtained in step b) with ammonium sulfate of 20% to 100% saturation, and dissolving the resulting precipitate in a buffer and dialyzing against the buffer;
- d) loading the product of step c) onto an anion exchange column, equilibrating the column with a buffer, and collecting the loading flow-through and the equilibrating flow-through, and dialyzing the collected loading flow-through and the equilibrating flow-through against the buffer;
- e) loading the product of step d) onto a cation exchange column, and equilibrating the column with a buffer, performing linear gradient elution with a sodium chloride solution based on the same buffer, collecting fractions of the eluate with heparinase activity, and optionally dialyzing the collected eluate against the buffer.
A method for producing heparinase, wherein the method comprises the steps of:
- a) culturing a Pseudomonas stutzeri strain deposited at the China Center for Type Culture Collection under the Accession Number CCTCC M 2017174 and collecting the cultured bacterial cells;
- b) resuspending and lysing the cells obtained in step a) in a buffer and collecting the supernatant after centrifugation;
- c) precipitating the supernatant obtained in step b) with ammonium sulfate of 20% to 100% saturation, and dissolving the resulting precipitate in a buffer and dialyzing against the buffer;
- d) loading the product of step c) onto an anion exchange column, and equilibrating the column with a buffer, performing linear gradient elution with a sodium chloride solution based on the same buffer, collecting fractions of the eluate with heparinase activity, and optionally dialyzing the collected eluate against the buffer.
A method for producing a low molecular weight heparin or an ultra-low molecular weight heparin, comprising the step of contacting the heparin with the recombinant heparinase comprising an amino acid sequence shown in SEQ ID NO:2.
Dependencies are inferred from the claim text.