TY - JOUR
T1 - A thermostable sugar-binding protein from the archaeon Pyrococcus horikoshii as a probe for the development of a stable fluorescence biosensor for diabetic patients
AU - Staiano, Maria
AU - Sapio, MariaRosaria
AU - Scognamiglio, Viviana
AU - Marabotti, Anna
AU - Facchiano, Angelo M.
AU - Bazzicalupo, Paolo
AU - Rossi, Mose'
AU - D'Auria, Sabato
PY - 2004/9
Y1 - 2004/9
N2 - 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.
AB - 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.
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M3 - Article
C2 - 15458346
AN - SCOPUS:5444238423
VL - 20
SP - 1572
EP - 1577
JO - Biotechnology Progress
JF - Biotechnology Progress
SN - 8756-7938
IS - 5
ER -