title: Development of a novel fluorescent protein construct by genetically fusing green fluorescent protein to the N-terminal of aspartate dehydrogenase.
abstract: 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-Asp) 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-Glu, the fluorescence signal was 2.7 times lower than that observed for l-Asp. In the present study, we describe two novelties: development of a genetically encoded fluorescent protein construct for monitoring of l-Asp 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-Asp. 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-Asp binding protein, we attempted to use the substrate specificity of aspartate dehydrogenase enzyme.
authors.affiliation: Department of Biochemistry, Faculty of Science, Ege University, Izmir, Turkey.
authors.forename: Canan
authors.initials: C
authors.lastname: Ozyurt
authors.forename: Serap
authors.initials: S
authors.lastname: Evran
authors.forename: Azmi
authors.initials: A
authors.lastname: Telefoncu
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D018698:Glutamic Acid / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism*; D020124:Thermotoga maritima / Q000201:enzymology
chemical_list: D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D000594:Amino Acid Oxidoreductases; C025745:aspartate dehydrogenase
keywords: Thermotoga maritima; biorecognition element; fluorescent protein