Expression in <i>Escherichia coli</i>, purification and characterization of heparinase I from <i>Flavobacterium heparinum</i>

Ernst, Steffen; Venkataraman, Ganesh; Winkler, Stefan; Godavarti, Ranga; Langer, Robert; Cl, Cooney; Sasisekharan, Ram

doi:10.1042/bj3150589

Cited by 63 publications

(57 citation statements)

References 54 publications

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“…Both the total and soluble protein expression levels achieved were unsatisfactory, however, especially given our previous successes with recombinantly expressing other HSGAG-degrading enzymes cloned from F. heparinum (1,29,30). As has been the case for most of these enzymes, removal of their putative N-terminal signal sequences greatly facilitated the recombinant expression of soluble protein without compromising their respective specific activities.…”

Section: Molecular Cloning and Recombinant Expression Of The F Heparmentioning

confidence: 99%

The Heparin/Heparan Sulfate 2-O-Sulfatase from Flavobacterium heparinum

Myette

Shriver

Claycamp

et al. 2003

Journal of Biological Chemistry

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Heparan sulfate glycosaminoglycans are structurally complex polysaccharides critically engaged in a wide range of cell and tissue functions. Any structure-based approach to study their respective biological functions is facilitated by the use of select heparan sulfate glycosaminoglycan-degrading enzymes with unique substrate specificities. We recently reported of one such enzyme, the ⌬4,5-glycuronidase cloned from Flavobacterium heparinum and recombinantly expressed in Escherichia coli (Myette, J. R., Shriver, Z., Kiziltepe, T., McLean, M. W., Venkataraman, G., and Sasisekharan, R. (2002) Biochemistry 41, 7424 -7434). In this study, we likewise report the molecular cloning of the 2-O-sulfatase from the same bacterium and its recombinant expression as a soluble, highly active enzyme. At the protein level, the flavobacterial 2-O-sulfatase possesses considerable sequence homology to other members of a large sulfatase family, especially within its amino terminus, where the highly conserved sulfatase domain is located. Within this domain, we have identified by sequence homology the critical active site cysteine predicted to be chemically modified as a formylglycine in vivo. We also present a characterization of the biochemical properties of the enzyme as it relates to optimal in vitro reaction conditions and a kinetic description of its substrate specificity. In particular, we demonstrate that in addition to the fact that the enzyme exclusively hydrolyzes the sulfate at the 2-O-position of the uronic acid, it also exhibits a kinetic preference for highly sulfated glucosamines within each disaccharide unit, especially those possessing a 6-O-sulfate. The sulfatase also displays a clear kinetic preference for disaccharides with ␤134 linkages but is able, nevertheless, to hydrolyze unsaturated, 2-O-sulfated chondroitin disaccharides. Finally, we describe the substrate-product relationship of the 2-O-sulfatase to the ⌬4,5-glycuronidase and the analytical value of using both of these enzymes in tandem for elucidating heparin/heparan sulfate composition.

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Section: Molecular Cloning and Recombinant Expression Of The F Heparmentioning

confidence: 99%

The Heparin/Heparan Sulfate 2-O-Sulfatase from Flavobacterium heparinum

Myette

Shriver

Claycamp

et al. 2003

Journal of Biological Chemistry

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“…Recombinant Hep I and III were expressed and purified to homogeneity, as described (9,10). The enzymes were incubated with endotoxin removal resin (Associates of Cape Cod) to ensure its removal.…”

Section: Methodsmentioning

confidence: 99%

Tumor cell surface heparan sulfate as cryptic promoters or inhibitors of tumor growth and metastasis

Liu

Shriver²,

Venkataraman³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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Heparan sulfate glycosaminoglycans, present at the cell surface and in the extracellular matrix that surrounds cells, are important mediators of complex biological processes. Furthermore, it is now apparent that cells dynamically regulate the structure of their heparan sulfate ''coat'' to differentially regulate extracellular signals. In the present study, the importance of sequence information contained within tumor cell-surface heparan sulfate was investigated. Herein, we demonstrate that the heparan sulfate glycosaminoglycan coat present on tumor cells contains bioactive sequences that impinge on tumor-cell growth and metastasis. Importantly, we find that growth promoting as well as growth inhibiting sequences are contained within the polysaccharide coat. Furthermore, we find that the dynamic balance between these distinct polysaccharide populations regulates specific intracellular signal-transduction pathways. This study not only provides a framework for the development of polysaccharide-based anti-cancer molecules but also underscores the importance of understanding a cell's polysaccharide array in addition to its protein complement, to understand how genotype translates to phenotype in this postgenomic age.

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“…An inclusion body of r-heparinase from F. heparinum was also formed in E. coli by the addition of a high concentration of IPTG. 12,29) Although it was solubilized by treatment with guanidine hydrochloride and refolded, the increase in activity was not very great. The production of rheparinase by an E. coli recombinant harboring pHEP8 requires no IPTG induction, and the enzyme production barely exceeded that by B. circulans HpT298 as described in Results.…”

Section: Discussionmentioning

confidence: 99%