Abstract
Green fluorescent protein (GFP) is the most widespread fluorescent reporter for cellular localization and interaction of proteins. Because GFP itself is not the protein purification tag, protein purification is generally carried out with the aid of additional affinity tags. We have recently engineered a “multifunctional GFP” (mfGFP), a variant of enhanced GFP (EGFP), in which multiple affinity tags are inserted in tandem into an internal loop of EGFP. The mfGFP can be used as a fluorescent reporter and an affinity tag, and is compatible with various expression systems in prokaryotic and eukaryotic cells. Herein, we describe detailed procedures for the expression and purification of mfGFP fusion proteins in mammalian cells. A method for tandem affinity purification using two different tags within mfGFP is also described.
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References
Shimomura O (2009) Discovery of green fluorescent protein (GFP) (Nobel Lecture). Angew Chem Int Ed Engl 48:5590–5602
Chalfie M (2009) GFP: lighting up life (Nobel Lecture). Angew Chem Int Ed Engl 48:5603–5611
Tsien RY (2009) Constructing and exploiting the fluorescent protein paintbox (Nobel Lecture). Angew Chem Int Ed Engl 48:5612–5626
Einhauer A, Jungbauer A (2001) The FLAG peptide, a versatile fusion tag for the purification of recombinant proteins. J Biochem Biophys Methods 49:455–465
Abedi MR, Caponigro G et al (1998) Green fluorescent protein as a scaffold for intracellular presentation of peptides. Nucleic Acids Res 26:623–630
Paramban RI, Bugos RC et al (2004) Engineering green fluorescent protein as a dual functional tag. Biotechnol Bioeng 86:687–697
Zhong JQ, Freyzon Y et al (2004) Enhanced detection sensitivity using a novel solid-phase incorporated affinity fluorescent protein biosensor. Biomol Eng 21:67–72
Keefe AD, Wilson DS et al (2001) One-step purification of recombinant proteins using a nanomolar-affinity streptavidin-binding peptide, the SBP-Tag. Protein Expr Purif 23:440–446
Kobayashi T, Morone N et al (2008) Engineering a novel multifunctional green fluorescent protein tag for a wide variety of protein research. PLoS One 3:e3822
Kobayashi T, Murayama T (2009) Cell cycle-dependent microtubule-based dynamic transport of cytoplasmic dynein in mammalian cells. PLoS One 4:e7827
Piston DW, Kremers GJ (2007) Fluorescent protein FRET: the good, the bad and the ugly. Trends Biochem Sci 32:407–414
Puig O, Caspary F et al (2001) The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24:218–229
O’Gorman S, Fox DT et al (1991) Recombinase-mediated gene activation and site-specific integration in mammalian cells. Science 251:1351–1355
Schmidt TG, Skerra A (2007) The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins. Nat Protoc 2:1528–1535
Getz EB, Xiao M et al (1999) A comparison between the sulfhydryl reductants tris(2-carboxyethyl)phosphine and dithiothreitol for use in protein biochemistry. Anal Biochem 273:73–80
Acknowledgments
This work was supported in part by grants-in-aid for scientific research from the Japan Society for the Promotion of Science and the Japan Science and Technology Agency (SENTAN).
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Murayama, T., Kobayashi, T. (2014). Purification of Recombinant Proteins with a Multifunctional GFP Tag. In: Giannone, R., Dykstra, A. (eds) Protein Affinity Tags. Methods in Molecular Biology, vol 1177. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1034-2_12
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DOI: https://doi.org/10.1007/978-1-4939-1034-2_12
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-1034-2
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