PREPARATION OF BIOTINYLATED BETA-GALACTOSIDASE CONJUGATES FOR COMPETITIVE-BINDING ASSAYS BY POSTTRANSLATIONAL MODIFICATION OF RECOMBINANT PROTEINS

A biotinylated beta-galactosidase conjugate prepared by the posttranslational modification of a recombinant fusion protein was used in the development of a heterogeneous binding assay for biotin. This conjugate was biotinylated at a predetermined location on a polypeptide tag attached to the N-terminus of beta-galactosidase. A kinetic study using the purified conjugate showed that the genetically engineered biotinylated beta-galactosidase has a slightly smaller K-m for o-nitrophenyl beta-D-galactopyranoside than that found for the native enzyme. The biotinylated beta-galactosidase was used to develop heterogeneous binding assays for biotin using both avidin and streptavidin-coated beads. Dose-response curves obtained by employing two different batches of biotinylated beta-galactosidase prepared 4 weeks apart were essentially identical, indicating the potential advantage of long-term assay reproducibility attainable through the use of recombinant enzyme-analyte conjugates. This is made possible by the inherent specificity of the process of recombinant protein expression and posttranslational modification in Escherichia coli, resulting in the highly reproducible preparation of conjugates.