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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Fluorescent and Luminescent Fusion Proteins for Detection ofAmyloid Beta Peptide Localization and AggregationKenji Usui 1,2,3 , Masayasu Mie 3 , Takashi Andou 3 , Naoki Sugimoto 1,2 ,Hisakazu Mihara 3 , and Eiry Kobatake 31 Faculty of Frontiers of Innovative Research in Science and Technology (FIRST), KonanUniversity, Kobe, 650-0047, Japan; 2 Frontier Institute for Biomolecular EngineeringResearch (FIBER), Konan University, Kobe, 650-0047, Japan; 3 Graduate School ofBioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, 226-8501, JapanIntroductionIn Alzheimer's disease (AD), the amyloid beta peptide (Aβ) forms fibrillar aggregatesknown as amyloid fibrils. This amyloid fibril is the principal component of extracellulardeposits which are regarded as causative agents. However, how Aβ can aggregate in vivoand how the generated aggregates affect the disease development, are still in question.Since recent studies have implicated Aβ and its ability to self-assemble as key factors in thepathogenesis of AD [1], understanding the behaviors of Aβ in vivo, such as where Aβ canaccumulate and what structure (monomers, oligomers or fibrils) Aβ adopts at eachlocalized point, is a promising clue to these mysteries. From this point of view,development of molecules that can bind to Aβ and can provide some output signalsdepending on Aβ structural states would be required. In this study, the fusion protein,consisting of Aβ sequence, and fluorescent and luminescent proteins was constructed toanalyze Aβ localization and aggregation in internal and external cells.Results and DiscussionFirst of all, the fusion protein, Y-Aβ-L (Figure 1), consisting of Aβ sequence, andfluorescent and luminescent proteins (EYFP; enhanced yellow fluorescent protein, andhRluc; humanized Renilla luciferase) at both termini of the Aβ sequence was designed. TheAβ sequence in the fusion protein was employed to give some affinities andco-aggregations with Aβ. Fusion of EYFP and hRluc with the Aβ sequence was applied toallow detecting Aβ localization and monitoring conformational changes. According torecent papers [2,3], it was expected that EYFP fluorescence and/or hRluc luminescencecould be increased or decreased when the conformation of the fusion protein was varied.Then, expression vectors for the fusion proteins were constructed and the protein wasexpressed in E. coli and was purified. Using the protein, we conducted co-aggregationexperiments by monitoring fluorescent and luminescent changes during incubation with Aβ.After the 20 hr incubation, Y-Aβ-L with Aβ showed that its luminescence was significantlydecreased but that there were little changes in fluorescence. This implied that the detectionsystem for Aβ localization and aggregation could be achieved with luminescence givingAβ conformational information and with fluorescence contributing Aβ localizationinformation. In addition, Y-Aβ-L alone gave no fluorescence and no luminescence after theincubation and dynamic light scattering data showed that hydrodynamic radius of Y-Aβ-Lalone was increased with the incubation. TEM images also supported that Y-Aβ-L aloneaggregated to amorphous form. These results implied that Y-Aβ-L alone could aggregateby itself and that this aggregation seemed more intense than wild type Aβs. This mightreduce the background fluorescence of Y-Aβ-L alone in the detection of Aβ localization.Fig. 1. Structure of the fusion protein.488