An Enzyme-Labeled Protein Polymer Bearing Pendent Haptens

Abstract

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 beta-casein. The resultant trinitrophenylated beta-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 beta-casein-TNB conjugates. The resultant AP-labeled beta-casein-bearing pendent TNB moieties (AP-betaCT) showed comparable specific activity with native AP. It was found that only the AP-betaCT 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-betaCT was demonstrated by competitive immunoassays for trinitrophenol (TNP) and trinitrotoluene (TNT), with detection limits of 0.99 microg/L and 0.18 microg/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.

Antibody microarrays for high-throughput, multianalyte analysis

“…There is considerable evidence that PTMs are modified in diseases such as breast cancer and can be used to distinguish cancer stages or for monitoring therapeutic response [15]. Conventional ELISA analysis employing 96-well plates has been widely used for studies on PTMs [62][63][64][65][66].…”

Orthogonal Enzymatic Reactions for the Assembly of Proteins at Electrode Addresses

“…While transglutaminases are common in mammalian tissue, the microbial version of this enzyme offers advantages for technical applications. − Importantly, the formation of the amide linkage occurs at mTG’s active site which confers selectivity to the reaction. The selectivity of mTG has been used for the site-selective modification of proteins with small molecules (e.g., labels and haptens), − the generation of heterodimeric proteins, − the immobilization of proteins, , and the coupling of proteins to other biological polymers (e.g., carbohydrates − and DNA) that have been suitably modified to serve as a substrate for mTG.…”