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.
- Enhanced green fluorescent protein (EGFP)
- Hydrolytic enzymes
ASJC Scopus subject areas
- Molecular Biology