Engineering of a monomeric fluorescent protein AsGFP499 and its applications in a dual translocation and transcription assay.
The tetrameric green fluorescent protein AsGFP(499) 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 degrees 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)-AsGFP(499) 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.
Tasdemir
Aynur
A
Axxam SpA, Via Olgettina 58, 20132 Milan, Italy.
Khan
Farid
F
Jowitt
Thomas A
TA
Iuzzolino
Lucia
L
Lohmer
Stefan
S
Corazza
Sabrina
S
Schmidt
Thomas J
TJ
D000042:Absorption; D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D002850:Chromatography, Gel; D019281:Dimerization; D015195:Drug Design; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050778:NFATC Transcription Factors / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D020836:Protein Structure, Quaternary; D021381:Protein Transport; D012615:Sea Anemones / Q000378:metabolism*; D016415:Sequence Alignment; D015398:Signal Transduction / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014158:Transcription, Genetic
D050505:Mutant Proteins; D050778:NFATC Transcription Factors; D049452:Green Fluorescent Proteins; D008156:Luciferases