A new methodology for the preparation of enzyme-labeled protein polymers bearing pendent haptens was developed through the combination of chemical modification and posttranslational protein modification catalyzed by microbial transglutaminase (MTG). As a model hapten, trinitrobenzene (TNB) was chosen and chemically conjugated with the accessible Lys residues of β-casein. The resultant trinitrophenylated β-casein was further modified with formaldehyde to render the residual Lys residues inert toward self-cross-linking by MTG. Escherichia coli alkaline phosphatase (AP), comprising a specific peptide tag carrying a MTG-reactive Lys residue, was then conjugated to the Gln residues in β-casein-TNB conjugates. The resultant AP-labeled β-casein-bearing pendent TNB moieties (AP-βCT) showed comparable specific activity with native AP. It was found that only the AP-βCT with a sufficient number of pendent TNBs are capable of binding to a surface adsorbed with anti-TNP and anti-TNT antibodies, indicating the presence of polyvalent interactions. The utility of AP-βCT was demonstrated by competitive immunoassays for trinitrophenol (TNP) and trinitrotoluene (TNT), with detection limits of 0.99 μg/L and 0.18 μg/L, respectively. The present study demonstrates the potential of dual labeling of protein scaffolds by chemical and enzymatic protein manipulation to create a new proteinaceous architecture.
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