Katie White and Tao Uttamapinant / REPRINTED WITH PERMISSION FROM Proc Natl Acad Sci U S A, 107:10914-19, 2010.

The paper

C. Uttamapinant et al., “A fluorophore ligase for site-specific protein labeling inside living cells,” Proc Natl Acad Sci USA, 107:10914-19, 2010. Free F1000 Evaluation

The finding

Visualizing proteins is crucial for understanding normal cell function, yet current labeling methods are limited. Despite their remarkable specificity, GFP-type proteins are too bulky, often exceeding the size of their protein targets—thus interfering with their function and trafficking—and the only small peptide label in use is toxic to cells. To address this, Alice Ting from the Massachusetts Institute of Technology and colleagues developed an enzyme-mediated intracellular protein label that’s small, highly specific, and not toxic.

The feat

The researchers created a one-step labeling process whereby an engineered E. coli ligase attaches a blue fluorophore to a short amino acid tail engineered to...

The novelty

Intriguingly, the technique offers “the possibility to selectively label subpopulations of the proteins” in various cell compartments, write Kasper Runager and Faculty Member David Rubenstein in their review. The researchers engineered the ligase gene to also express a short protein tag found exclusively either in the membrane, cytosol, or nucleus, tricking the cell into trafficking the ligase to that location for target protein labeling.

The future

The team is currently trying to improve the ligase for compartments such as the endoplasmic reticulum (ER) and Golgi apparatus, whose oxidizing environments prevent the ligase function.

F1000 evaluators: M. Golynskiy and B. Seelig (Univ Minnesota) • J. Grunewald (Genomics Inst of Novartis Research Foundation) • J. Scheinost and R. Oliveira (Univ Oxford) • K. Runager and D. Rubenstein (UNC–Chapel Hill) • B.R. Oakley (Ohio State Univ)

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