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.
Affinity tag Green fluorescent protein (GFP) Immobilized metal affinity chromatography (IMAC) Multifunctional GFP (mfGFP) Protein complex Protein purification Streptavidin binding peptide (SBP)
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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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