Temporally controlled fluorescence labeling is a cinch with the SNAP tag, a fluorophore moiety designed to covalently attach to a fusion protein containing a 20-kDa variant of AGT (alkylguanine DNA alkyltransferase). When it comes to studying endocytosis and recycling of cell surface proteins, however, the tight grip of the SNAP tag is a blessing and a curse: unlike antibody labeling of cargo proteins, there's no chance of the fluorophore separating from its target; by the same token, one cannot remove the label from proteins remaining on the cell surface in order to selectively follow those that have been internalized. The solution, as shown by Cole and Donaldson in a recent article in ACS Chemical Biology, lies in modifying the SNAP tag to incorporate a cleavable disulfide bond. After AGT-containing cell surface proteins are labeled by the modified SNAP tag and then incubated to permit internalization, the reducing agent tris (2-carboxyethyl) phosphine (TCEP) is added to cleave off SNAP tags attached to proteins exposed on the cell surface. Importantly, TCEP is not membrane permeable, nor can it access disulfide bonds within cell surface proteins. Mere seconds after TCEP addition, the cell surface-associated fluorescence dissipates, giving the observer a clean view of tagged proteins in the endosomal compartments. To quantify uptake of a labeled receptor, one compares the total fluorescence before TCEP treatment with the signal remaining afterward, an improvement on existing endocytosis procedures that assay loss of signal on the cell surface (as measured by flow cytometry of antibody-labeled cell surface proteins). Biochemical assays are also possible, by fluorescence in-gel detection of lysates prepared from TCEP-treated cells. Although the authors’ experiments focus on G protein-coupled receptors, they point out the method is applicable to tracking endocytosis and recycling of any cell surface protein. Thanks to the range of possible fluorophores for SNAP tags, dual color imaging with other fluorescently labeled proteins is straight-forward. Together, these characteristics make releasable SNAP tags a simple, flexible tool to monitor endosomal dynamics.
Cole & Donaldson. Releasable SNAP-tag probes for studying endocytosis and recycling. ACS Chem Biol. [Epub ahead of print, January 13, 2012; doi: 10.1021/ cb2004252].MAGE On Center Stage
An article in one of the debut issues of the journal ACS Synthetic Biology presents an ambitious application of multiplex automated genome engineering. MAGE, as it is known, was developed by George Church's group as a method to simultaneously modify multiple loci in a bacterial genome. The goal is to engineer entire networks or biosynthetic pathways at once, which MAGE achieves through the use of oligo-mediated allelic replacement in an Escherichia coli strain expressing the λ-Red recombination machinery. In their earlier publication, Church and colleagues describe using degenerate oligos to explore the sequence space of 24 genes related to lycopene biosynthesis. Many other applications become possible if multiple well-defined oligos are introduced simultaneously, and it is in the new publication that Church and coworkers extend MAGE to wide-scale tagging of multiple enzyme pathway components for bulk affinity purification. In this case, the goal was cell-free protein synthesis, a process that works best when using defined-component systems such as PURE (protein synthesis using recombinant elements). The expense of individually purifying the translational machinery from E. coli is daunting, so the authors used MAGE to His tag multiple PURE components in a single strain. The result is a small collection of strains from which the entire PURE catalog can be isolated, an approach the authors call e(nsemble)PURE. In total, ePURE comprises six different strains, each containing four to eight His-tagged proteins, plus a strain for purification of the ribosome. Insertion efficiencies for the His tags ranged from 0.2% to 11.2%, but with cells tolerating 100+ MAGE cycles, even low-efficiency modifications had ample opportunity to arise. For in vitro translation, the authors pooled the nickel column eluates from the six strains expressing tagged translational factors, combining them with the affinity-purified ribosomes and necessary cofactors/ substrates. Though initial results were underwhelming, respectable activity was achievable by supplementing the mix with four translational factors that were poorly expressed in the ePURE strains. The authors point out that optimization by fine tuning the expression levels of individual factors is only a few MAGE steps away, and have made the ePURE strains freely available without restriction. This new ensemble will be music to the ears of those coping with the expense of purchasing individual PURE components; more importantly, this extension of MAGE to parallel protein modification will doubtless inspire much future improvisation on the theme of single-pot, multi-enzyme catalysis.
Wang et al. Multiplexed in vivo His-tagging of enzyme pathways for in vitro single-pot multi-enzyme catalysis. ACS Synth Biol. [Epub ahead of print, January 30, 2012; doi: 10.1021/ sb3000029].