Jennifer C. Lewis

Peer Reviewed: Applications of Reporter Genes

Reporter proteins can be used in bioanalytical methods to produce signals indicating the presence of a target analyte.

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

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.

Fluorescence binding assay for a small peptide based on a GFP fusion protein

Abstract A fluorescence binding assay was developed for a small peptide based on a fusion protein between the peptide and the green fluorescent protein, GFP. The assay employs genetic engineering methods to prepare the analyte-label (peptide–GFP) conjugate as a fusion protein in order to produce a one-to-one, homogenous population of labeled-peptide. Specifically, a plasmid was constructed in which the C-terminus of a model octapeptide was fused to the N-terminus of GFP. Following expression of the octapeptide–GFP fusion protein in Escherichia coli , an immunoassay was developed based on sequential binding of the free octapeptide and labeled-octapeptide to an anti-octapeptide antibody immobilized on a solid surface. The naturally fluorescent protein acts as a label to provide sensitive detection for peptides. To our knowledge, this is the first time that GFP has been used as a quantitative label in a fusion protein to develop a quantitative assay for a peptide analyte.