In Vitro Fluorescent Analysis of Preprotein Import into Chloroplasts

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  • KWANCHANOK Sattasuk
    The United Graduate School of Agricultural Sciences, Ehime University
  • INOUE Hitoshi
    The United Graduate School of Agricultural Sciences, Ehime University
  • AKITA Mitsuru
    The United Graduate School of Agricultural Sciences, Ehime University Faculty of Agriculture, Ehime University Satellite Business Venture, Ehime University

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  • <i>In Vitro</i>Fluorescent Analysis of Preprotein Import into Chloroplasts

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Abstract

Despite recent progress in fluorescence techniques employed to observe protein localization in living cells, the in vitro chloroplastic protein transport assay remains a useful tool for determining the destinations of proteins. Although an in vitro synthesized, radiolabeled precursor protein is frequently used as the transport substrate, we have developed a transport assay system with a non-radiolabeled precursor protein that carries an epitope tag and is overexpressed in Escherichia coli. Thus, a transported protein can be detected by immunoblotting (Inoue et al., Plant Physiol. Biochem., 46, 541–549 (2008)). Here, we propose another in vitro protein transport system that combines fluorescence techniques. We attempted to use two types of precursors: a green fluorescent protein (GFP)-fused precursor and a fluorescent dye-labeled one. Both were successfully imported into chloroplasts. However, the fluorescent dye-labeled precursor was more advantageous than the GFP-fused precursor in the in vitro system.

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