Improving the cost effectiveness of enhanced green fluorescent protein production using recombinant Escherichia coli BL21 (DE3): Decreasing the expression inducer concentration.

Green fluorescent protein (GFP) is a globular protein used as biosensor and biomarker in medical and industrial fields. However, due to the expensive production costs of expressing proteins using high-cost inducers like isopropyl-ß-d-1-thiogalactopyranoside (IPTG), the number of GFP applications are still scarce. This work studied the production of enhanced GFP (EGFP) using Escherichia coli BL21 (DE3) [pLysS; pET28(a)], aiming to increase its yield and reduce costs. First, the influence of agitation rate, induction time, and concentration of IPTG in the production of EGFP was evaluated, but only the first two parameters were significant. Subsequently, aiming to reduce costs related to the use of inducer, the IPTG concentration (0.005, 0.010, and 0.025 mM) was decreased and, interestingly, the production levels were maintained or increased. These results show that a proper choice of production conditions, particularly through the decrease of inducer concentration, is effective to reduce the upstream production costs and guarantee high EGFP expression.

Reversible and irreversible fluorescence activity of the Enhanced Green Fluorescent Protein in pH: Insights for the development of pH-biosensors.

Enhanced Green Fluorescent Protein (EGFP) is a biomolecule with intense and natural fluorescence, with biological and medical applications. Although widely used as a biomarker in research, its application as a biosensor is limited by the lack of in-depth knowledge regarding its structure and behavior in adverse conditions. This study is focused on addressing this need by evaluating EGFP activity and structure at different pH using three-dimensional fluorescence, circular dichroism and small-angle X-ray scattering. The focus was on the reversibility of the process to gain insights for the development of biocompatible pH-biosensors. EGFP was highly stable at alkaline pH and quenched from neutral-to-acidic pH. Above pH 6.0, the fluorescence loss was almost completely reversible on return to neutral pH, but only partially reversible from pH 5.0 to 2.0. This work updates the knowledge regarding EGFP behavior in pH by accounting for the recent data on its structure. Hence, it is evident that EGFP presents the required properties for use as natural, biocompatible and environmentally friendly neutral to acidic pH-biosensors.

Revealing a new fluorescence peak of the enhanced green fluorescent protein using three-dimensional fluorescence spectroscopy.

Fluorescent proteins have many applications as biomarkers and biosensors in the medical and biological fields. Their success was largely supported by modifications of the first isolated fluorescent protein, the wild-type Green Fluorescent Protein (wtGFP), which allowed the development of improved variants such as the Enhanced GFP (EGFP). The first reports on EGFP indicated that the protein presented a single form and fluorescence peak, in contrast to the two conformations observed in wtGFP. However, after experimental determination of the crystalline structure of EGFP, two conformations were found, generating questions regarding the relationship between EGFP structure and its spectral characteristics. To resolve the controversy, this study evaluated EGFP 3D fluorescence spectra at lower wavelengths and under distinct conditions (different concentrations, pH and temperatures), revealing the existence of a second fluorescence peak for this protein. It was possible to confirm that the new peak was not a reflection of the intrinsic fluorescence of proteins or an artefact from the 3D fluorescence spectroscopy. It was also shown that the second peak is pH dependent, sensitive to high temperatures and linearly related to EGFP concentration, confirming a direct relationship between the new fluorescence peak and EGFP protein structure. In addition to the revelation of the new EGFP fluorescence peak, this study demonstrated that 3D fluorescence can be used as powerful technique in the discovery of other elusive fluorophores.