| Summary: | Previously, the binding specificities of three antibodies, Jel42, Jel44, and Jel323, specific for histidine-containing protein, HPr, of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system (PTS) were characterized by competition SPRIA using a panel of site-specific mutants of HPr, and the data was used collectively to define the epitopes of each of the antibodies (Sharma et al., 1991; Sharma, 1992). Subsequently, the crystal structure of the Jel42 Fab/HPr complex confirmed the epitope mapped by mutagenesis (Prasad et al., 1993). In order to study the paratope of Jel42 by site-directed mutagenesis, a gene encoding the structural gene of the scFv fragment was constructed for expression in Escherichia coli. The gene was assembled from synthetic oligonucleotides and designed to maximize unique restriction endonuclease sites as well as codons frequently found in highly expressed proteins of E. coli. SPRIA assays and competitions cannot be used to determine the antibody binding constants, norcan they be used to evaluate antibody fragment specificity without a fragment-specific secondary antibody. A new fluorescence polarization assay was developed to determine the binding constants and specificity, as well as the thermodynamic parameters of binding, for these antibodies and their fragments. Site-specific cysteine mutants of HPr labelled with thiol-specific fluorescein 5-maleimide were used as fluorescent tracers in the binding studies; labelled Arg17Cys HPr was used for Jel42 and Jel44 but was unsuitable for Jel323 because of fluorescence quenching, so labelled Phe2Cys was used for those studies. All antibody fragments bound with affinities comparable to their respective parent antibodies with Kd values all in the low nM range: Jel42, 2.8 ± 1.3 nM; Jel42 Fab, 3.3 ± 0.2 nM; Jel42 scFv, 2.7 ± 2.0 nM; Jel44, 5.1 ± 0.9 nM; Jel44 Fab, 5.8 ± 0.3 nM; Jel323, 5.2 ± 0.5 nM; and Jel323 Fab, 6.0 ± 0.7 nM. The binding specificity of each antibody, as determined by SPRIA, was confirmed using this assay, and the antibody fragments were found to share the same specificity as their respective parent antibody. Neither Jel42 nor Jel323 was affected by changes in ionic strength from 30 to 230 mM NaCl, but Jel44 binding decreased 2-3 fold with salt increases over this range. This assay was also used to investigate the thermodynamic parameters of binding of the three antibodies and the Jel42 fragments.
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