Nature Biotechnology 23, 1308 - 1314 (2005)
Published online: 11 September 2005; | doi:10.1038/nbt1136
Mammalian cell−based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinityBrent R Martin1, 2, Ben N G Giepmans1, 3, 4, Stephen R Adams1
& Roger Y Tsien1, 5, 61
Department of Pharmacology, University of California, San Diego, 9500 Gilman Dr., La Jolla, California, 92093-0647, USA. 2
Biomedical Sciences Graduate Program, University of California, San Diego, 9500 Gilman Dr., La Jolla, California, 92093-0647, USA. 3
Department of Neurosciences, University of California, San Diego, 9500 Gilman Dr., La Jolla, California, 92093-0647, USA. 4
National Center for Microscopy and Imaging Research, University of California, San Diego, 9500 Gilman Dr., La Jolla, California, 92093-0647, USA. 5
Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Dr., La Jolla, California
92093-0647, USA. 6
Howard Hughes Medical Institute, University of California, San Diego, 9500 Gilman Dr., La Jolla, California, 92093-0647, USA.
Correspondence should be addressed to Roger Y Tsien rtsien@ucsd.edu Membrane-permeant biarsenical dyes such as FlAsH and ReAsH fluoresce upon binding to genetically encoded tetracysteine motifs expressed in living cells1,
2, yet spontaneous nonspecific background staining can prevent detection of weakly expressed or dilute proteins2,
3. If the affinity of the tetracysteine peptide could be increased, more stringent dithiol washes should increase the contrast between specific and nonspecific staining. Residues surrounding the tetracysteine motif were randomized and fused to GFP, retrovirally transduced into mammalian cells and iteratively sorted by fluorescence-activated cell sorting for high FRET from GFP to ReAsH in the presence of increasing concentrations of dithiol competitors. The selected sequences show higher fluorescence quantum yields and markedly improved dithiol resistance, culminating in a >20-fold increase in contrast. The selected tetracysteine sequences, HRWCCPGCCKTF and FLNCCPGCCMEP, maintain their enhanced properties as fusions to either terminus of GFP or directly to  -actin. These improved biarsenical-tetracysteine motifs should enable detection of a much broader spectrum of cellular proteins.
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