Development of a GABA Enzyme for Biosensor Applications

Abstract As the most important inhibitory neurotransmitter in the braina detailed understanding of the implications of gamma aminobutyric acidGABArelease remains elusiveThe measurement of GABA concentrations is a difficult processMicrodialysis is the current standard for GABA sampling in the brains of freely moving animalsbut suffers from low temporal resolution and the need for labor intensive analysis methodsBy contrastthe direct sensing of GABAby modalities including biosensorswould provide second by second temporal resolutionwithout the need for additional postanalysisHoweverbiosensors and other monitoring devicesrequire an enzyme to process the analyte of interestThe state of the art for the enzymatic conversion of GABA into a transducible signal is the sequential activity of multiple enzymes or antibodies entrapped within nanoparticlesFor brain and systemic GABA sensing applicationsa single GABA oxidase enzyme is necessaryNo such oxidase enzyme for GABA is currently availableThis proposal addresses this problem by designing a single GABA specific oxidase enzyme for use as the biorecognition element that is suitable for fabrication of a GABA biosensorDuring Phase I we identifiedisolatedpurifiedclonedcrystallized and modeled the active site structure ofNMethylaminobutyrate oxidaseMGOXWe set up a directed evolution systemKJin which there will only be growth if GABA is available as a nitrogen sourceand we demonstrated that the preliminary MGOX enzyme can be used on a biosensor to detect GABAFor Phase II of this projectthere are two major problems that must be solvedModification of MGOX to increase its kcat at physiological pHpHandModification of MGOX to increase sensitivity to detect physiologically relevant GABA concentrationsPinnacle will team with an interdisciplinary group of two leading scientists at the University of KansasProfessor Mark Richteris an expert in protein engineering and protein foldingand DrPhilip Gaois the Director of the Protein Production Core FacilityBy the end of Phase IItwo commercially available products will be availableFirsta GABA biosensor for real time measurement of physiologically relevant levels of GABA in the brain for preclinical animal models and seconda GABA oxidase enzyme for use in a variety of diagnostic and point of care devices