An enzyme-labeled protein polymer bearing pendent haptens.
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.
Tominaga
Jo
J
Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan.
Kamiya
Noriho
N
Goto
Masahiro
M
D000469:Alkaline Phosphatase / Q000737:chemistry*; D000906:Antibodies / Q000737:chemistry; D002364:Caseins / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000201:enzymology; D005973:Glutamine / Q000737:chemistry; D006241:Haptens / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D011506:Proteins / Q000737:chemistry; D013499:Surface Properties; D011503:Transglutaminases / Q000737:chemistry; D014301:Trinitrobenzenes / Q000032:analysis / Q000737:chemistry*
D000906:Antibodies; D002364:Caseins; D006241:Haptens; D011108:Polymers; D011506:Proteins; D014301:Trinitrobenzenes; D005973:Glutamine; D011503:Transglutaminases; D000469:Alkaline Phosphatase; D008239:Lysine