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A Thermostable Sugar‐Binding Protein from the Archaeon Pyrococcus horikoshii as a Probe for the Development of a Stable Fluorescence Biosensor for Diabetic Patients
Author(s) -
Staiano Maria,
Sapio MariaRosaria,
Scognamiglio Viviana,
Marabotti Anna,
Facchiano Angelo M.,
Bazzicalupo Paolo,
Rossi Mose',
d'Auria Sabato
Publication year - 2004
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp049857g
Subject(s) - circular dichroism , biosensor , pyrococcus horikoshii , chemistry , recombinant dna , chromatography , fluorescence spectroscopy , biochemistry , affinity chromatography , size exclusion chromatography , sodium dodecyl sulfate , fluorescence , enzyme , physics , quantum mechanics , gene
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.