Development of an assay for β-lactam hydrolysis using the pH-dependence of enhanced green fluorescent protein

• Published in: Analytical biochemistry 2002-10, Vol.309 (2), p.224-231
• Main Authors: Puckett, Libby G, Lewis, Jennifer C, Bachas, Leonidas G, Daunert, Sylvia
• Format: Article
• Language: English
• Subjects: Aequorin - chemistry; Ampicillin - metabolism; Anti-Bacterial Agents - metabolism; beta-Lactamases - metabolism; Calibration; Enhanced green fluorescent protein (EGFP); Enzyme Inhibitors - pharmacology; Escherichia coli - metabolism; Fluorometry - methods; Green Fluorescent Proteins; Hydrogen-Ion Concentration; Hydrolysis; Hydrolytic enzymes; Kinetics; Luminescent Proteins - chemistry; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Plasmids; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Space life sciences; Sulbactam; Sulbactam - pharmacology; β-Lactamase
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/S0003-2697(02)00304-4

An assay has been developed utilizing the pH-dependent fluorescence of enhanced green fluorescent protein (EGFP). This photoprotein allows for the study of kinetic properties of hydrolytic enzymes based on the production of protons. As a model system, β-lactamase, a well-characterized enzyme responsible for antibiotic resistance in many bacteria, was used. More specifically, EGFP and β-lactamase were genetically fused using overlap extension PCR and incorporated into a bacterial expression vector. The vector was subsequently transformed into Escherichia coli, and the fusion protein was expressed and purified. β-Lactamase catalyzes the hydrolysis of the β-lactam ring of ampicillin. This causes a decrease in the local pH, which in turn changes the spectral properties of EGFP. This property was utilized to perform enzyme kinetic studies on the new fusion protein as well as on the β-lactamase inhibitor, sulbactam. The assay can be used to evaluate substrates and inhibitors of β-lactamase in a format that should be amenable to high-throughput screening.