Engineering of a monomeric fluorescent protein AsGFP499 and its applications in a dual translocation and transcription assay

Aynur Tasdemir, Farid Khan, Thomas A. Jowitt, Lucia Iuzzolino, Stefan Lohmer, Sabrina Corazza, Thomas J. Schmidt

    Research output: Contribution to journalArticlepeer-review


    The tetrameric green fluorescent protein AsGFP499 from the sea anemone Anemonia sulcata was converted into a dimeric and monomeric protein by site-directed mutagenesis. The protein was engineered without prior knowledge of its crystal structure based on a sequence alignment of multiple proteins belonging to the GFP-family. Crucial residues for oligomerisation of AsGFP 499 were predicted and selected for mutation. By introduction of a single site mutation (S103K) the A/B subunit was disrupted whereas two substitutions were necessary to separate the A/C subunit (T159K/F173E). This method can be applied as a general predictive method for designing monomeric proteins from multimeric fluorescent proteins. The maturation temperature was optimised to 37°C by a combination of Site-directed and random mutagenesis. In cell-based assays, the NFATc1A (nuclear factor of activated T-cells, subtype 1, isoform A)-AsGFP499 chimera formed massive cytoplasmic aggregates in HeLa cells, which prevented the shuttling of NFATc1A into the nucleus and consequentially its transcriptional activity. In contrast, the cells expressing the NFATc1A in fusion with our engineered dimeric and monomeric fluorescent mutants were homogeneously distributed throughout the cytoplasm, making these stable cell lines functional in both translocation and transcriptonal assays. This new dual cellular assay will allow the screening and discovery of new drugs that target NFAT cellular processes. © The Author 2008. Published by Oxford University Press. All rights reserved.
    Original languageEnglish
    Pages (from-to)613-622
    Number of pages9
    JournalProtein Engineering, Design and Selection
    Issue number10
    Publication statusPublished - Oct 2008


    • AsGFP499
    • Fluorescent
    • GFP
    • Monomeric
    • NFATc1


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