Development of a novel fluorescent protein construct by genetically fusing green fluorescent protein to the N ‐terminal of aspartate dehydrogenase

We developed a fluorescent protein construct by genetically fusing green fluorescent protein (GFP) to aspartate dehydrogenase from Thermotoga maritima . The fusion protein was cloned, heterologously expressed in Escherichia coli cells, and purified by Ni‐chelate affinity chromatography. It was then introduced into a measurement cuvette to monitor its fluorescence signal. Aspartate dehydrogenase functioned as the biorecognition element, and aspartate‐induced conformational change was converted to a fluorescence signal by GFP . The recombinant protein responded to l ‐aspartate (l ‐ A sp) linearly within the concentration range of 1–50 mM, and it was capable of giving a fluorescence signal in 1 Min. Although a linear response was also observed for l ‐ G lu, the fluorescence signal was 2.7 times lower than that observed for l ‐ A sp. In the present study, we describe two novelties: development of a genetically encoded fluorescent protein construct for monitoring of l ‐ A sp in vitro , and employment of aspartate dehydrogenase scaffold as a biorecognition element. A few genetically encoded amino‐acid biosensors have been described in the literature, but to our knowledge, a protein has not been constructed solely for determination of l ‐ A sp. Periplasmic ligand binding proteins offer high binding affinity in the micromolar range, and they are frequently used as biorecognition elements. Instead of choosing a periplasmic l ‐ A sp binding protein, we attempted to use the substrate specificity of aspartate dehydrogenase enzyme.