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46. Preparation of biotinylated beta-galactosidase conjugates for competitive binding assays by posttranslational modification of recombinant proteins

Author

Witkowski, Allan, Kindy, Mark S., Daunert, Sylvia, and Bachas, Leonidas G.

Subjects
Post-translational modification -- Research, Proteins -- Synthesis, Recombinant proteins -- Research, Chemistry
Abstract

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.sub.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.

Published
1995

49. Enzyme-linked immunosorbent assay for an octapeptide based on a genetically enineered fusion protein

Author

Witkowski, Allan, Daunert, Sylvia, Kindy, Mark S., and Bachas, Leonidas G.

Subjects
Enzyme-linked immunosorbent assay -- Methods, Peptides -- Research, Gene fusion -- Usage, Chemistry
Abstract

Traditional chemical means of preparing enzymeligand conjugates for use in enzyme-linked immunosorbent assays (ELISAs) lead to the production of multisubstituted enzyme-ligand conjugates with a high degree of variability in the site of ligand attachment. A genetically engineered fusion protein was prepared in order to investigate the feasibility of controlled production of conjugates for use in ELISAs. Specifically, a synthetic octapeptide was fused with bacterial alkaline phosphatase. The resulting enzymepeptide conjugate is monosubstituted (one peptide per subunit), has a single site of attachment, and results in assays with good response characteristics. The use of such fusion proteins, which combine small analyte peptides with enzyme labels, can lead to a new approach to improved assays for numerous biomolecules, including peptide pharmaceuticals, neurotransmitters, hormones, cell surface antigens, etc., The viability of enzyme-linked conjugates for the development of enzyme- linked immunosorbent assays was tested. Experiments involving the genetic fusion of a synthetic octapeptide with bacterial alkaline phosphatase were conducted. A monosubstituted enzyme-peptide conjugate with one attachment site was produced. The advantages of the approach in terms of selectivity trade-off and batch variability were demonstrated.

Published
1993

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