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    Systematic Analysis of FKBP Inducible Degradation Domain Tagging Strategies for the Human Malaria Parasite Plasmodium falciparum

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    Author
    de Azevedo, MF; Gilson, PR; Gabriel, HB; Simoes, RF; Angrisano, F; Baum, J; Crabb, BS; Wunderlich, G
    Date
    2012-07-16
    Source Title
    PLOS ONE
    Publisher
    PUBLIC LIBRARY SCIENCE
    University of Melbourne Author/s
    Crabb, Brendan; BAUM, JACOB; Angrisano, Fiona
    Affiliation
    Medical Biology (W.E.H.I.)
    Metadata
    Show full item record
    Document Type
    Journal Article
    Citations
    de Azevedo, M. F., Gilson, P. R., Gabriel, H. B., Simoes, R. F., Angrisano, F., Baum, J., Crabb, B. S. & Wunderlich, G. (2012). Systematic Analysis of FKBP Inducible Degradation Domain Tagging Strategies for the Human Malaria Parasite Plasmodium falciparum. PLOS ONE, 7 (7), https://doi.org/10.1371/journal.pone.0040981.
    Access Status
    Access this item via the Open Access location
    URI
    http://hdl.handle.net/11343/33067
    DOI
    10.1371/journal.pone.0040981
    Open Access at PMC
    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3397994
    Description

    C1 - Journal Articles Refereed

    Abstract
    Targeted regulation of protein levels is an important tool to gain insights into the role of proteins essential to cell function and development. In recent years, a method based on mutated forms of the human FKBP12 has been established and used to great effect in various cell types to explore protein function. The mutated FKBP protein, referred to as destabilization domain (DD) tag when fused with a native protein at the N- or C-terminus targets the protein for proteosomal degradation. Regulated expression is achieved via addition of a compound, Shld-1, that stabilizes the protein and prevents degradation. A limited number of studies have used this system to provide powerful insight into protein function in the human malaria parasite Plasmodium falciparum. In order to better understand the DD inducible system in P. falciparum, we studied the effect of Shld-1 on parasite growth, demonstrating that although development is not impaired, it is delayed, requiring the appropriate controls for phenotype interpretation. We explored the quantified regulation of reporter Green Fluorescent Protein (GFP) and luciferase constructs fused to three DD variants in parasite cells either via transient or stable transfection. The regulation obtained with the original FKBP derived DD domain was compared to two triple mutants DD24 and DD29, which had been described to provide better regulation for C-terminal tagging in other cell types. When cloned to the C-terminal of reporter proteins, DD24 provided the strongest regulation allowing reporter activity to be reduced to lower levels than DD and to restore the activity of stabilised proteins to higher levels than DD29. Importantly, DD24 has not previously been applied to regulate proteins in P. falciparum. The possibility of regulating an exported protein was addressed by targeting the Ring-Infected Erythrocyte Surface Antigen (RESA) at its C-terminus. The tagged protein demonstrated an important modulation of its expression.
    Keywords
    Medical Parasitology; Infectious Diseases

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