Abstract
This protocol describes an efficient method to site-specifically label cell-surface or purified proteins with chemical probes in two steps: probe incorporation mediated by enzymes (PRIME) followed by chelation-assisted copper-catalyzed azide-alkyne cycloaddition (CuAAC). In the PRIME step, Escherichia coli lipoic acid ligase (LplA) site-specifically attaches a picolyl azide (pAz) derivative to a 13-aa recognition sequence that has been genetically fused onto the protein of interest. Proteins bearing pAz are chemoselectively derivatized with an alkyne-probe conjugate by chelation-assisted CuAAC in the second step. We describe herein the optimized protocols to synthesize pAz to perform PRIME labeling and to achieve CuAAC derivatization of pAz on live cells, fixed cells and purified proteins. Reagent preparations, including synthesis of pAz probes and expression of LplA, take 12 d, whereas the procedure for performing site-specific pAz ligation and CuAAC on cells or on purified proteins takes 40 min–3 h.
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Change history
04 December 2013
In the version of this article initially published, Katharine A. White, Scott Grecian, Scott Clarke and Kyle R. Gee were not present in the author list. The error has been corrected in the HTML and PDF versions of the article. The author contributions section and competing financial interests statement have also been modified to include information relevant to these authors.
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Acknowledgements
We thank Ting laboratory members who have contributed to PRIME-related protocols, particularly M. Fernández-Suárez. We also thank A. Karunakaran (University of California, San Francisco (UCSF)) and R.D. Vale (UCSF) for LAP-kinesin K560 and kinesin K420 proteins, C. Garner (Stanford University) for the neuron fixative recipe and M.G. Finn (Scripps Research Institute) for helpful advice on CuAAC. Funding was provided by the US National Institutes of Health (R01 GM072670) and the Dreyfus Foundation.
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Contributions
C.U., M.I.S., D.S.L., J.Z.Y., K.A.W., S.G., S.C., K.R.G. and A.Y.T. developed protocols. C.U. contributed all the data. C.U., M.I.S. and A.Y.T. wrote the paper.
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Competing interests
A.Y.T. holds a patent (publication number US8137925 B2) related to methods that use lipoic acid ligase for specific protein labeling. Life Technologies has applied for a patent related to picolyl azide reagents.
Supplementary information
Supplementary Figure 1
Estimation of cell-surface labeling yield by streptavidin gel shift assay. HEK cells expressing LAP-tagged cyan fluorescent protein fused to the transmembrane domain of the platelet-derived growth factor receptor (LAP-CFP-TM1) were labeled with 200 μM picolyl azide for 20 min using exogenously supplied W37VLplA, then by live-cell CuAAC for 5 min with 20 μM biotin-alkyne and 50 μM CuSO4. Cells were then lysed under hypotonic conditions and incubated with ∼10 μM streptavidin for 30 min at room temperature. The reaction mixture was analyzed on 12% SDS-PAGE without boiling the samples, to preserve biotin-streptavidin binding. CFP-containing species were visualized by in-gel CFP fluorescence. The shift in the mobility of a population of LAP-CFP-TM upon streptavidin addition gives the yield of biotin-alkyne modification on LAP-CFP-TM. Negative controls with streptavidin omitted (lane 1) or with AP-CFP-TM2 replacing LAP-CFP-TM (lanes 3-4) showed no change in mobility. We previously estimated that ∼50% of TM fusion proteins (such as LAP-CFP-TM) are located at the cell surface, with the rest in the ER, Golgi, or endosomes. Therefore, if ∼38% of total LAP-CFP-TM is biotinylated by our standard labeling protocol (average of two independent replicates), we estimate that ∼76% of its cell surface pool is labeled. (PDF 571 kb)
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Uttamapinant, C., Sanchez, M., Liu, D. et al. Site-specific protein labeling using PRIME and chelation-assisted click chemistry. Nat Protoc 8, 1620–1634 (2013). https://doi.org/10.1038/nprot.2013.096
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DOI: https://doi.org/10.1038/nprot.2013.096
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