Abstract
Fluorescent tagging of proteins has become a critical step in optical analysis of protein function in vitro and in living cells. Here we describe a two-tag system for expression and isolation of a protein of interest from Escherichia coli and subsequent site-specific fluorescent labeling with Sfp phosphopantetheinyl transferase (Sfp synthase). In the example presented, adenoviral protein E3-14.7 K (E14.7) was expressed as a tripartite fusion protein with a fluorophore-targeting peptide tag and a chitin-binding domain. This system allows for rapid isolation of the recombinant fusion protein from crude bacterial cell lysate via a single chitin column. Sfp synthase-mediated labeling with fluorophore conjugated to coenzyme A-SH (CoA-SH) resulted in covalent attachment of a fluorescent dye to a specific residue of the peptide tag via a phosphopantetheinyl linker. The fluorescently labeled E14.7 fusion protein was analyzed with a fluorescence imager and subsequently transfected into mammalian cells for imaging with a fluorescence microscope.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Wang, H., Chen, X. (2008) Site-specifically modified fusion proteins for molecular imaging. Front Biosci 13, 1716–1732.
Chen I., Ting A. Y. (2005) Site-specific labeling of proteins with small molecules in live cells. Curr Opin Biotechnol 16, 35–40.
Tsien, R. Y. (1998) The green fluorescent protein. Annu Rev Biochem 67, 509–544.
Griffin B. A., Adams, S. R., Tsien, R. Y. (1998) Specific covalent labeling of recombinant protein molecules inside live cells. Science 10, 269–272.
Keppler, A., Gendreizig, S., Gronemeyer, T., Pick, H., Vogel, H., Johnsson, K. (2002) A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat Biotechnol 21, 86–89.
Yin, J., Lin, A. J., Golan, D. E., Walsh, C. T. (2006) Site-specific protein labeling by Sfp phosphopantetheinyl transferase. Nat Protoc 1, 280–285.
Zhou, Z., Cironi, P., Lin, A. J., Xu, Y., Hrvatin, S., Golan, D. E., et al. (2007) Genetically encoded short peptide tags for Orthogonal protein labeling by Sfp and Acps phosphopantetheinyl transferases. ACS Chem Biol 2, 337–346.
Gautier, A., Juillerat, A., Heinis, C., Corrêa, I. R., Kindermann, M., Beaufils, F., Johnsson, K. (2008) An engineered protein tag for multiprotein labeling in living cells. Chem Biol 15, 128–136.
Meyer, B. H., Segura, J., Martinez, K. L., Hovius, R., George, N., Johnsson, K., Vogel, H. (2006) FRET imaging reveals that functional neurokinin-1 receptors are monomeric and reside in membrane microdomains of live cells. Proc Natl Acad Sci USA 103, 2138–2143.
Yin, J., Lin, A. J., Buckett, P. D., Wessling-Resnick, M., Golan, D. E., Walsh, C. T. (2005) Single-cell FRET imaging of transferrin receptor trafficking dynamics by Sfp-catalyzed, site-specific protein labeling. Chem Biol 12, 999–1006.
Jacquier, V., Prummer, M., Segura, J.-M., Pick, H., Vogel, H. (2006) Visualizing odorant receptor trafficking in living cells down to the single-molecule level. Proc Natl Acad Sci USA 103, 14325–14330.
Yin, J., Straight, P. D., McLoughlin, S. M., Zhou, Z., Lin, A. J., Golan, D. E., et al. (2005) Genetically encoded short peptide tag for versatile protein labeling by Sfp phosphopantetheinyl transferase. Proc Natl Acad Sci USA 102, 15815–15820.
Schneider-Brachert, W., Tchikov, V., Merkel, O., Jakob, M., Hallas, C., et al. (2006) Inhibition of TNF receptor 1 internalization by adenovirus 14.7 K as a novel immune escape mechanism. J Clin Invest 116, 2901–2913.
Ferrandon, S., Sterzenbach, T., Mersha, F., Xu, M.-Q. (2003) A single surface tryptophan in the chitin-binding domain from Bacillus circulans chitinase A1 plays a pivotal role in binding chitin. Biochim Biophys Acta 1621, 31–40.
Evans, T. C., Jr., Benner, J., Xu, M.-Q. (1998) Semisynthesis of cytotoxic proteins using a modified protein splicing element. Protein Sci 7, 2256–2264.
Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248–254.
Spector, D., Goldman, R. (eds.) (2004). Live Cell Imaging: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Acknowledgments
The authors thank New England Biolabs and Donald G. Comb, Jim Ellard, Richard Roberts, Ted Davis, Salvatore Russello, Thomas C. Evans, Jr., Williams Jack, Jack Benner, Sriharsa Pradhan, Christopher Provost for their encouragement and valuable suggestions. We thank the Organic Division for peptide and fluorescent substrate synthesis. We also thank Drs. Christopher Walsh and Jun Yin for kindly providing pET29-Sfp for expression and purification of Sfp synthase.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Zhang, A. et al. (2011). Fluorescent Site-Specific Labeling of Escherichia coli Expressed Proteins with Sfp Phosphopantetheinyl Transferase. In: Evans, Jr., T., Xu, MQ. (eds) Heterologous Gene Expression in E.coli. Methods in Molecular Biology, vol 705. Humana Press. https://doi.org/10.1007/978-1-61737-967-3_18
Download citation
DOI: https://doi.org/10.1007/978-1-61737-967-3_18
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61737-966-6
Online ISBN: 978-1-61737-967-3
eBook Packages: Springer Protocols