A novel reagentless sensing system for measuring glucose based on the galactose/glucose-binding protein

The galactose/glucose-binding protein (GBP) is synthesized in the cytoplasm of Escherichia coli in a precursor form and exported into the periplasmic space upon cleavage of a 23-amino-acid leader sequence. GBP binds galactose and glucose in a highly specific manner. The ligand induces a hinge motion in GBP and the esultant protein conformational change constitutes the basi s of the sensing system. The mglB gene, which codes for GBP, was isolated f rom the chromosome off. coli using the polymerase chain reaction (PCR). Sin ce wild-type GBP lacks cysteines in its structure, introducing this amino a cid by site-directed mutagenesis ensures single-label attachment at specifi c sites with a sulfhydro-specific fluorescent probe. Site-directed mutagene sis by overlap extension PCR was performed to prepare three different mutan ts to introduce a single cysteine residue at positions 148, 152, and 182. S ince these residues are not involved in ligand binding and since they are l ocated at the edge of the binding cleft, they experience a significant chan ge in environment upon binding of galactose or glucose. The sensing system strategy is based on the fluorescence changes of the probe as the protein u ndergoes a structural change on binding. In this work a reagentless sensing system has been rationally designed that can detect submicromolar concentr ations of glucose. The calibration plots have a linear working range of thr ee orders of magnitude. Although the system can sense galactose as well, th is epimer is not a potential interfering substance since its concentration in blood is negligible. (C) 2001 Academic Press.