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PEDS Advance Access published online on August 1, 2008
Protein Engineering Design and Selection, doi:10.1093/protein/gzn040
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

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

Aynur Tasdemir1,4, Farid Khan2, Thomas A. Jowitt3, Lucia Iuzzolino1, Stefan Lohmer1, Sabrina Corazza1,5 and Thomas J. Schmidt4,5

1Axxam SpA, Via Olgettina 58, 20132 Milan, Italy 2Lumophore Limited, 20 Oakleigh Avenue, Timperley, Cheshire WA15 6QT, UK 3Faculty of Life Sciences, The University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK 4 Institute for Pharmaceutical Biology und Phytochemistry (IPBP), Westfälische Wilhelms-Universtität, Münster, Hittorfstraße 56, D-48149 Münster, Germany

5 To whom correspondence should be addressed. E-mail: thomschm{at}uni-muenster.de (T.J.S.)/sabrina.corazza.sc{at}axxam.com (S.C)

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 AsGFP499 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.

Keywords: AsGFP499/fluorescent/GFP/monomeric/NFATc1

Received April 21, 2008; revised June 27, 2008; accepted July 2, 2008.


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