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AssigneeIbex Technologies Ltd.
InventorsHongsheng Su, Francoise Blain, Clark Bennett, Kangfu Gu, Joseph Zimmermann, Roy Musil
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The present invention describes the isolation and sequence of genes from Flavobacterium heparinum encoding heparin and heparan sulfate degrading enzymes, heparinase II and heparinase III (EC 4.2.2.8). It further describes a method of expressing and an expression for heparinases I, II and III using a modified ribosome binding region derived from a promoter from glycosaminoglycan lyase genes of F. heparinum. Also, a multi-step protein purification method incorporating cell disruption, cation exchange chromatography, affinity chromatography and hydroxylapatite chromatography is outlined. Antibodies against a post-translational modification moiety common to Flavobacterium heparinum proteins and a method to obtain antibodies specific to these moieties and to the amino acid sequences of heparinases I, II and III are described.
Isolated, recombinant non-glycosylated Flavobacterium heparinum heparinase II in substantially pure form.
Isolated and purified recombinant non-glycosylated heparinase II comprising the amino acid sequence of SEQ ID NO:2.
Isolated and purified heparinase II comprising the amino acid sequence of SEQ ID NO:2 as expressed in E. coli cells.
Isolated and purified Flavobacterium heparinum heparinase III wherein the amino terminus is not a modified pyroglutamate residue.
Isolated and purified heparinase III comprising the amino acid sequence of SEQ ID NO:4 wherein the amino terminus is not a modified pyroglutamate residue.
Isolated and purified heparinase III comprising the amino acid sequence of SEQ ID NO:4 as expressed in E. coli cells.
Dependencies are inferred from the claim text.