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 resultant protein conformational change constitutes the basis of the sensing system. The mglB gene, which codes for GBP, was isolated from the chromosome of E. coli using the polymerase chain reaction (PCR). Since wild-type GBP lacks cysteines in its structure, introducing this amino acid by site-directed mutagenesis ensures single-label attachment at specific sites with a sulfhydro-specific fluorescent probe. Site-directed mutagenesis by overlap extension PCR was performed to prepare three different mutants to introduce a single cysteine residue at positions 148, 152, and 182. Since these residues are not involved in ligand binding and since they are located at the edge of the binding cleft, they experience a significant change in environment upon binding of galactose or glucose. The sensing system strategy is based on the fluorescence changes of the probe as the protein undergoes a structural change on binding. In this work a reagentless sensing system has been rationally designed that can detect submicromolar concentrations of glucose. The calibration plots have a linear working range of three orders of magnitude. Although the system can sense galactose as well, this epimer is not a potential interfering substance since its concentration in blood is negligible.
Salins
L L
LL
Department of Chemistry, University of Kentucky, Lexington, Kentucky, 40506-0055, USA.
Ware
R A
RA
Ensor
C M
CM
Daunert
S
S
D001483:Base Sequence; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D005947:Glucose / Q000032:analysis*; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D033902:Periplasmic Binding Proteins; D016133:Polymerase Chain Reaction; D011487:Protein Conformation
D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D017931:DNA Primers; D005456:Fluorescent Dyes; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; C023550:galactose-binding protein; D005947:Glucose