title: Purification of recombinant proteins with a multifunctional GFP tag.
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.
authors.affiliation: Department of Cellular and Molecular Pharmacology, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan, takashim@juntendo.ac.jp.
authors.forename: Takashi
authors.initials: T
authors.lastname: Murayama
authors.forename: Takuya
authors.initials: T
authors.lastname: Kobayashi
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D057809:HEK293 Cells; D006639:Histidine / Q000737:chemistry; D006801:Humans; D008967:Molecular Biology / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D002352:Carrier Proteins; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; C452700:streptavidin-binding peptide; D049452:Green Fluorescent Proteins; D006639:Histidine