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When imaging living cells, it is still a challenge to specifically and efficiently label the target protein, and to do so without disrupting protein function. Fluorescent protein fusions have been a popular means to achieve labeling, but they have their drawbacks. Although it's easy to create these fusions, according to David Golan of Harvard University, “The problem from an imaging point of view is that when you synthesize fluorescent protein inside the cell, the whole biosynthetic pathway becomes fluorescently labeled.” Small synthetic fluorescent probes, which have higher sensitivity and resolution for imaging studies, have been developed as alternatives, but their use is limited by a lack of methods to quickly and covalently label proteins with these probes.

Recently, Christopher Walsh's group developed a method for site-specific post-translational labeling of proteins in cell lysates with small molecules (Yin et al., 2004). They fused an 80-residue peptide carrier protein (PCP) to the target protein and then used the enzyme Sfp phosphopantetheinyl transferase to catalyze the covalent modification of a specific serine in PCP.

Now, in an article in Chemistry & Biology, Christopher Walsh, David Golan, Marianne Wessling-Resnick and colleagues have collaborated to extend this method to specifically label a cell-surface protein in living cells (Yin et al., 2005). They labeled a transferrin receptor–PCP fusion and its ligand, transferrin, with Alexa fluorophores, which allowed them to use fluorescence resonance energy transfer (FRET) imaging to follow trafficking of this receptor-ligand pair through the cell in real time. Using this method, they showed that transferrin is bound to the receptor throughout the endocytic and exocytic cycle, corrobo-rating prior biochemical studies.

According to Golan, this work shows “the power of a great method,” and the authors plan to use the same approach to study other trafficking pathways. There are also efforts underway to use this method to label intracellular proteins, potentially extending its applicability to many more fields.