Engineering and rapid selection of a low‐affinity glucose/galactose‐binding protein for a glucose biosensor

Abstract

Periplasmic expression screening is a selection technique used to enrich high-affinity proteins in Escherichia coli. We report using this screening method to rapidly select a mutated D-glucose/Dgalactose-binding protein (GGBP) having low affinity to glucose. Wild-type GGBP has an equilibrium dissociation constant of 0.2 mM and mediates the transport of glucose within the periplasm of E. coli. The protein undergoes a large conformational change on binding glucose and, when labeled with an environmentally sensitive fluorophore, GGBP can relay glucose concentrations, making it of potential interest as a biosensor for diabetics. This use necessitates altering the glucose affinity of GGBP, bringing it into the physiologically relevant range for monitoring glucose in humans (1.7-33 mM). To accomplish this a focused library was constructed using structure-based site-saturation mutagenesis to randomize amino acids in the binding pocket of GGBP at or near direct H-bonding sites and screening the library within the bacterial periplasm. After selection, equilibrium dissociation constants were confirmed by glucose titration and fluorescence monitoring of purified mutants labeled site-specifically at E149C with the fluorophore IANBD (N,N9-dimethyl-N-(iodoacetyl)-N9-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene-diamine). The screening identified a single mutation A213R that lowers GGBP glucose affinity 5000-fold to 1 mM. Computational modeling suggested the large decrease in affinity was accomplished by the arginine side chain perturbing H-bonding and increasing the entropic barrier to the closed conformation. Overall, these experiments demonstrate the ability of structure-based sitesaturation mutagenesis and periplasmic expression screening to discover low-affinity GGBP mutants having potential utility for measuring glucose in humans.Keywords: GGBP; glucose/galactose-binding protein; low affinity; structure-based site-saturation mutagenesis; screening; focused library Abbreviations: GGBP, glucose/galactose-binding protein; IANBD, N,N9-dimethyl-N-(iodoacetyl)-N9-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylenediamine; wt, wild type; ORF, open reading frame; LOD, limit of detection; DPBS, Dulbecco's phosphate-buffered saline; PBS, phosphate-buffered saline.Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi

Ligand-Binding Proteins: Structure, Stability and Practical Application

“…Changing the affinity and specificity of ligand-binding proteins has been achieved by the re-engineering of the protein's ligand-binding site. Much work has been performed with PBP-based biosensors for sugar detection (e.g., glucose, ribose and other) (Amiss et al, 2007;Medintz & Deschamps, 2006;Vercillo et al, 2007). Utilization of the Escherichia coli D-galactose/Dglucose-binding protein as the sensitive element in the glucose biosensor is one of the most promising directions for continuous glucose monitoring (Deuchle et al, 2005;Shilton et al, 1996).…”

“…Utilization of the Escherichia coli D-galactose/Dglucose-binding protein as the sensitive element in the glucose biosensor is one of the most promising directions for continuous glucose monitoring (Deuchle et al, 2005;Shilton et al, 1996). In this case, the necessity to lower the affinity of GGBP to glucose should be taken into account in the development of methods to monitor the glucose level in human blood (Amiss et al, 2007). It is worth mentioning that the ligand-binding proteins represent a target for the development of antimicrobial agents.…”

Spectral characteristics of the mutant protein D-glucose/D-galactose-binding protein GGBP/H152C with an attached fluorescent dye BADAN: Influence of external conditions

“…Examples of such proteins include concanavalin A and the Dglucose/D-galactose-binding protein (GGBP). This application of GGBP is promising because the protein-glucose binding promotes a significant change in the tertiary structure of GGBP (Yesylevskyy et al, 2006;Sakaguchi-Mikami et al;2008, Saxl et al;, Amiss et al, 2007.…”

The role of water molecules in stereoselectivity of glucose/galactose-binding protein

“…Because of this property, PBPs are highly valued for industrial use as components of biosensor systems involving a fluorescent nanosensor456. In particular, they are studied as glucose-sensing elements that can be used in the development of a blood glucose self-monitoring system78. Glucose/galactose-binding protein (GGBP) is a good example of a PBP that can be used for development of a glucose biosensor protein.…”

“…To turn GGBP into a glucose sensor, many studies based on protein-engineering have been conducted7813, and one of the studies has actually shown that Asp14-mutated GGBPs (as a result of site-directed mutagenesis) have glucose-selective binding affinity under controlled experimental conditions14. However, the cause of the glucose-selective affinity in mutated GGBP was not explained.…”