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Vol. 15, Issue 12, 5616-5622, December 2004

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Short Tetracysteine Tags to -Tubulin Demonstrate the Significance of Small Labels for Live Cell Imaging

Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany

Submitted June 6, 2004; Revised September 13, 2004; Accepted September 23, 2004
Monitoring Editor: Ted Salmon

Genetically encoded tags are of fundamental importance for live cell imaging. We show that small tetracysteine (TetCys) tags can be highly advantageous for the functionality of the host protein compared with large fluorescent protein tags. One to three concatenated small TetCys tags as well as the large green fluorescent protein (GFP) were fused by integrative epitope tagging to the C terminus of -tubulin (Tub2) in the budding yeast Saccharomyces cerevisiae. The increasing tag size correlated with functional interference to the host protein. Tub2 tagged with either 1xTetCys (10 amino acids [aa]) or 2xTetCys (20 aa) was able to substitute Tub2 in haploid cells. In contrast, C-terminal tagging of Tub2 with 3xTetCys (29 aa) or with GFP (244 aa) resulted in nonviable haploid cells. Cells expressing Tub2-1xTetCys or Tub2-2xTetCys were stained with FlAsH, which selectively binds to the TetCys-tag. The stained cells displayed dynamic FlAsH-labeled microtubules and low cellular background fluorescence. The presented approach to tag open reading frames (ORFs) at their native loci with very small TetCys-tags and the subsequent visualization of the tagged proteins in vivo can be extended in principle to any ORF in S. cerevisiae.

Abbreviations used: aa, amino acid; EDT, 1,2-ethanedithiol; FlAsH-EDT₂, fluorescein arsenical helix binder, bis-EDT adduct (4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein); FP, fluorescent protein; GFP, green fluorescent protein; ORF, open reading frame; TetCys, tetracysteine.

^* Corresponding author. E-mail address: sjakobs{at}gwdg.de.

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