Production of heparanase constructs suitable for nuclear magnetic resonance and drug discovery studies

Silvia Mosulén, Leticia Ortí, Esperanza Bas, Rodrigo J. Carbajo, Antonio Pineda-Lucena

Research output: Contribution to journalArticle

7 Scopus citations

Abstract

Heparanase is an endo-β-D-glucosidase capable of specifically degrading heparan sulphate, one of the main components of the extracellular matrix. This 65 kDa polypeptide is implicated in cancer processes such as tumour formation, angiogenesis and metastasis, making it a very attractive target in antitumour treatments. Structure-based approaches to find inhibitors of heparanase have been historically hampered by the lack of success in crystallizing the protein. With the aim to undertake the NMR structural characterisation of heparanase, we have designed and produced, using recombinant methods, smaller constructs of heparanase containing the catalytically active glutamic acids and the two binding sites for heparan sulphate. An extensive range of expression and purification conditions were evaluated to alleviate the intrinsic low solubility and aggregation propensity of heparanase, allowing the obtention of the enzyme in milligram quantities, both unlabelled and 15N-labelled for NMR studies. Using the smallest of the designed constructs and applying NMR and SPR methodologies, we have demonstrated that known inhibitors of heparanase bind to this construct specifically and selectively with KD values in the range of those reported for human heparanase, validating it for future drug discovery projects focused on the identification of novel inhibitors of this enzyme.

Original languageEnglish (US)
Pages (from-to)151-160
Number of pages10
JournalBiopolymers
Volume95
Issue number2
DOIs
StatePublished - Feb 1 2011
Externally publishedYes

Keywords

  • NMR
  • SPR
  • drug discovery
  • expression
  • heparan sulphate
  • heparanase
  • purification

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Biomaterials
  • Organic Chemistry

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