First vertebrates fluorescence protein UnaG: bacterial expression, purification and characterization

Many fluorescence proteins and variants, including green fluorescent protein which was discovered in 1998, have been identified and used in scientific studies. In 2013, the first fluorescence protein UnaG, originating from the vertebrate, was identified and its properties were demonstrated. In the present study, the UnaG protein was produced by fusion with TolAIII in Escherichia coli and its characterization was performed. In this study, the UnaG protein was purified by histidine affinity chromatography from Escherichia coli and, the presence and purity of the protein was demonstrated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and western blotting; also, further characterization studies were performed by UV/ViS, Fluorescence and CD spectroscopy. According to Far-Uv CD results, the dominant secondary structure in the protein was determined as anti-parallel beta-sheet. Thermal fluorescence studies have shown that the protein remains stable even at higher than room temperature. In the titration studies performed against 6,5 nM protein, it was determined that the protein reached saturation at 11,67 nM un-conjugated bilirubin concentration. It is possible to carry out different studies in the field of medicine and bioengineering by using UnaG, the first vertebrate source fluorescence protein that shows high affinity and specificity against un-conjugated bilirubin.