title: A protein switch sensing system for the quantification of sulfate.
abstract: Protein engineering has generated versatile methods and technologies that have been instrumental in advancements in the fields of sensing, therapeutics, and diagnostics. Herein, we demonstrate the employment of rational design to engineer a unique bioluminescence-based protein switch. A fusion protein switch combines two totally unrelated proteins, with distinct characteristics, in a manner such that the function of one protein is dependent on another. Herein we report a protein switch sensing system by insertion of the sulfate-binding protein (SBP) into the structure of the photoprotein aequorin (AEQ). In the presence of sulfate, SBP undergoes a conformational change bringing the two segments of AEQ together, "turning on" bioluminescence in a dose-dependent fashion, thus allowing quantitative detection of sulfate. A calibration plot was obtained by correlating the amount of bioluminescence generated with the concentration of sulfate present. The switch demonstrated selectivity and reproducibility, and a detection limit of 1.6×10(-4)M for sulfate. Moreover, the sensing system was validated by performing sulfate detection in clinical and environmental samples, such as, serum, urine, and tap water. The detection limits and working ranges in all three samples fall within the average normal/recommended sulfate levels in the respective matrices.
authors: Department of Chemistry, University of Kentucky, Lexington, KY 40506-0055, USA.
authors: Krystal Teasley
authors: KT
authors: Hamorsky
authors: Charles Mark
authors: CM
authors: Ensor
authors: Patrizia
authors: P
authors: Pasini
authors: Sylvia
authors: S
authors: Daunert
mesh_terms: D000331:Aequorin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D015374:Biosensing Techniques / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008163:Luminescent Measurements / Q000379:methods; D008958:Models, Molecular; D011487:Protein Conformation; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013431:Sulfates / Q000032:analysis*
chemical_list: D017931:DNA Primers; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D013431:Sulfates; C489040:sbp protein, E coli; D000331:Aequorin