Expression of the carbohydrate recognition domain of FimH and development of a competitive binding assay.
Uropathogenic Escherichia coli (UPEC) is the primary cause of urinary tract infections (UTIs). In the first step of this infective process, the virulence factor FimH located on type 1 pili allows UPEC to specifically adhere to oligosaccharides, which are part of glycoproteins on the urinary bladder mucosa. This initial step prevents the clearance of E. coli from the urinary tract and enables the invasion of the host cells. Because FimH antagonists can block this interaction, they exhibit a promising therapeutic potential as anti-infectives. For the evaluation of their binding properties, a reliable, target-based affinity assay is required. Here, we describe the expression and purification of the carbohydrate recognition domain of FimH (FimH-CRD) as well as the development of a competitive binding assay. FimH-CRD linked with a thrombin cleavage site to a 6His-tag is recombinantly expressed and purified by affinity chromatography. For the evaluation of FimH antagonists, a cell-free binding assay based on the interaction of a biotinylated polyacrylamide glycopolymer with the FimH-CRD was developed. Complexation of the biotinylated glycopolymer with streptavidin coupled to horseradish peroxidase allows the quantification of the binding properties of FimH antagonists. The assay format was optimized and validated by a comparison with affinity data from reported assays.
Rabbani
Said
S
Institute of Molecular Pharmacy, Pharmacenter, University of Basel, Switzerland Klingelbergstrasse 50, 4056 Basel, Switzerland.
Jiang
Xiaohua
X
Schwardt
Oliver
O
Ernst
Beat
B
D000180:Acrylic Resins / Q000737:chemistry; D018830:Adhesins, Escherichia coli / Q000235:genetics / Q000378:metabolism; D001667:Binding, Competitive; D003124:Colorimetry / Q000379:methods*; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D036781:Fimbriae Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D011108:Polymers / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000099166:Polyacrylamides
D000180:Acrylic Resins; D018830:Adhesins, Escherichia coli; D004121:Dimethyl Sulfoxide; D004492:Edetic Acid; D036781:Fimbriae Proteins; D006639:Histidine; D009842:Oligopeptides; D011108:Polymers; D011994:Recombinant Proteins; D000099166:Polyacrylamides; C471213:His-His-His-His-His-His; C058769:fimH protein, E coli