A thermostable sugar-binding protein from the archaeon Pyrococcus horikoshii as a probe for the development of a stable fluorescence biosensor for diabetic patients

Maria Staiano, MariaRosaria Sapio, Viviana Scognamiglio, Anna Marabotti, Angelo M. Facchiano, Paolo Bazzicalupo, Mose' Rossi, Sabato D'Auria

Research output: Contribution to journalArticlepeer-review

Abstract

In this work is presented the first attempt to develop an innovative ultrastable protein-based biosensor for blood glucose detections. The gene of a putative thermostable sugar-binding protein has been cloned and expressed in E. coli. The recombinant protein has been purified to homogeneity by thermoprecipitation and affinity chromatography steps. The recombinant protein is a monomer with an apparent molecular weight of 55,000 as judged by gel filtration and sodium dodecyl sulfate polyacrylamide gel eletrophoresis. Circular dichroism experiments showed that the protein possesses a secondary structure content rich in α-helices and β-structures and that the protein is highly stable as investigated in the range of temperature between 20 and 95°C. Fluorescence spectroscopy experiments demonstrated that the recombinant protein binds glucose with a dissociation constant of about 10 mM, a concentration of sugar very close to the concentration of glucose present in the human blood. A docking simulation on the modeled structure of the protein confirms its ability to bind glucose and proposes possible modifications to improve the affinity for glucose and/or its detection. The obtained results suggest the use of the protein as a probe for a stable glucose biosensor.

Original languageEnglish
Pages (from-to)1572-1577
Number of pages6
JournalBiotechnology Progress
Volume20
Issue number5
Publication statusPublished - Sep 2004

ASJC Scopus subject areas

  • Food Science
  • Biotechnology
  • Microbiology

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