Engineering of ligand specificity of periplasmic binding protein for glucose sensing.
A novel glucose-sensing molecule was created based on galactose/glucose-binding protein (GGBP). GGBP mutants at Asp14, a residue interacting with the 4th hydroxyl group of the sugar molecule, were constructed by mutagenesis to improve the ligand specificity of GGBP. The autofluorescence-based analysis of the binding abilities of these engineered GGBPs showed that the GGBP mutants Asp14Asn and Asp14Glu bound only to glucose in a concentration-dependent manner, without being affected by the presence of galactose. The Phe16Ala mutation, which leads to an increase in the K (d) value toward glucose, was then introduced into these two glucose-specific mutant GGBPs. One of the constructed GGBP double-mutants, Asp14Glu/Phe16Ala, had a glucose specificity with a K(d) value of 3.9 mM, which makes it suitable for use in the measurement of the physiological glucose concentration. Our results demonstrate that it is possible to construct a GGBP which specifically recognizes glucose and has a higher K(d) value and use it as a molecular recognition element of blood glucose monitoring systems by combining two different mutations based on the 3D structure of GGBP.
Sakaguchi-Mikami
Akane
A
Department of Biotechnology, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.
Taneoka
Atsushi
A
Yamoto
Rie
R
Ferri
Stefano
S
Sode
Koji
K
D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites; D015374:Biosensing Techniques / Q000379:methods*; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D005690:Galactose / Q000378:metabolism; D005818:Genetic Engineering; D005947:Glucose / Q000378:metabolism*; D008024:Ligands; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism; D033902:Periplasmic Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D010649:Phenylalanine / Q000235:genetics; D013050:Spectrometry, Fluorescence
D029968:Escherichia coli Proteins; D008024:Ligands; D009004:Monosaccharide Transport Proteins; D050505:Mutant Proteins; D033902:Periplasmic Binding Proteins; C509064:mglB protein, E coli; D001224:Aspartic Acid; D010649:Phenylalanine; D005947:Glucose; D005690:Galactose