SBIR-STTR Award

Heparin and related molecules are complex polysaccharides having numerous pharmaceutical applications: as anticoagulant, antithrombotic and antilipemic agents as well as for patients with osteoarthrosis. Also, there has been a growing interest in understanding the physiological functions of heparin like molecules in angiogenesis, cancer, glomerular permeability, neuron development, joint function, alzheimer's disease, etc. Heparin can be degraded enzymatically by heparinases which are very specific for defined residues in the polysaccharide chain. As such, the enzymes serve as powerful tools in studying the physiological role of heparin. Recently, the enzymes themselves have shown a potential for pharmaceutical and diagnostic applications; heparinases have been shown to be potent inhibitors of neovascularization. There has been no report of cloning any of the heparinases, other than heparinase I from Flavobacterium heparinum. This organism produces three heparinases--heparinase I, II and III. The absence of pure heparinases has limited clinical use of these enzymes. The overall goal of this work is to clone and produce heparinases II and III by recombinant DNA technology. This will allow catalytically pure heparinases to be available in large amounts - without contamination of related enzyme activities that occur in the materials currently produced commercially. PROPOSED COMMERCIAL APPLICATION: Cloning of the heparinase II and III genes will make these enzymes commercially available for several applications: which include, pre-clinical evaluation of heparinase III as a potential therapeutic agent, for the production of defined low molecular weight heparin fragments using heparinase II. Heparinases II and III are extensively used as important tools in cell and molecular biology research, and cloning these enzymes would facilitate the large scale production of these enzymes in a pure form.

Heparin and related molecules are complex polysaccharides having numerous pharmaceutical applications: as anti-coagulant, anti-thrombotic and anti- lipemic agents in several medical indications. Also, there is growing body of evidence that heparin plays a key role in neovascularization, breast, prostrate and other cancers, Alzheimer's and other neural diseases etc. Enzymes that degrade heparin-like molecules -heparinases are very specific for defined regions of the polysaccharide backbone. As such, heparinases, are essential reagents in studying structural, biochemical, physiological and pathological roles of heparin. Further, heparinases have shown potential for several diagnostic as well as therapeutic applications. Having cloned the heparinase genes from Flavobacterium heparinum during phase I studies, the phase II objective is to: 1) make very pure recombinant heparinases I. II and III commercially available. Recombinant production enormously facilitates contamination-free expression and cost- effective large scale production of heparinases. This also speeds the pending medical and clinical investigation of heparinases in several applications. 2) perform 'protein engineering' on heparinases to create 'designer' enzymes for different applications 3) develop a method to rapidly generate and separate low molecular weight heparins in a highly reproducible and cost-effective manner. 4) carry feasibility studies in the isolation and cloning of mammalian heparinases for therapeutic applications.Proposed commercial application:Large-scale production of heparinases will make these enzymes commercially available for several diagnostic as well as therapeutic applications. Further, heparinases are extensively used as important tools in Cell and molecular biology research.Thesaurus termsFlavobacteria, heparin lyase, peptide chemical synthesis, recombinant protein, synthetic enzyme heparin, method development, protein engineering high performance liquid chromatography, molecular cloning, protein purificationNational Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)