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

A biotinylated beta-galactosidase conjugate prepared by the posttransl ational modification of a recombinant fusion protein was used in the d evelopment of a heterogeneous binding assay for biotin. This conjugate was biotinylated at a predetermined location on a polypeptide tag att ached to the N-terminus of beta-galactosidase. A kinetic study using t he purified conjugate showed that the genetically engineered biotinyla ted beta-galactosidase has a slightly smaller K-m for o-nitrophenyl be ta-D-galactopyranoside than that found for the native enzyme. The biot inylated beta-galactosidase was used to develop heterogeneous binding assays for biotin using both avidin and streptavidin-coated beads. Dos e-response curves obtained by employing two different batches of bioti nylated beta-galactosidase prepared 4 weeks apart were essentially ide ntical, indicating the potential advantage of long-term assay reproduc ibility attainable through the use of recombinant enzyme-analyte conju gates. This is made possible by the inherent specificity of the proces s of recombinant protein expression and posttranslational modification in Escherichia coli, resulting in the highly reproducible preparation of conjugates.