Development and Applications of Enhanced Green Fluorescent Protein Mutants

Abstract

The introduction of several mutations resulted in the generation of improved mutants of the green fluorescent protein (GFP). A strong green (GFPsg25) and blue (BFPsg50) fluorescent protein, gave 50-fold-100-fold brighter fluorescence compared to wild-type GFP and BFP (Tyr66His), respectively, upon expression in mammalian cells. GFPsg25 and BFPsg50 have different excitation and emission maxima. This allows their use as an efficient dual-color tagging system and their independent detection in living cells.

The adenovirus type 5 E1B-55K oncoprotein is a highly active shuttle protein and shuttling is independent of E4orf6, p53 and Mdm2

“…pCMV-GFPsg25 and pCMV-BFPsg50 express versions of the green¯uorescent protein (GFP) emitting green or blue light, respectively, under the control of the CMV immediate early promoter (Stauber et al, 1998b). To generate the di erent pE1B-GFP expression plasmids, the E1B-55K coding regions of adenovirus type 5 or type 12 were ampli®ed by PCR using pXC15 (Logan et al, 1984) as templates and appropriate primers containing NheI-restriction sites.…”

Rev‐GFP transgenic lines for studies of nucleocytoplasmic transport in drosophila

“…Here we report another class of GFP-based nuclear transport reporter, Rev::GFPsg (Stauber et al, 1998a). The HIV viral protein Rev (regulator of viral expression) is responsible for the nuclear export of unspliced and partially spliced HIV mRNAs (Meyer and Malim, 1994;Pollard and Malim, 1998).…”

“…Because of this shuttling property, a Rev::GFPsg fusion has been constructed as a probe to determine nucleocytoplasmic trafficking properties in different mammalian cell types (Neumann et al, 2001). GFPsg is an enhanced form of GFP that is about 50 -100 times brighter than its progenitor (Stauber et al, 1998a). The Rev::GFPsg fusion protein behaves similarly as the untagged Rev protein (Stauber et al, 1998a).…”

“…GFPsg is an enhanced form of GFP that is about 50 -100 times brighter than its progenitor (Stauber et al, 1998a). The Rev::GFPsg fusion protein behaves similarly as the untagged Rev protein (Stauber et al, 1998a). It is localized to the nucleolus and is functionally equivalent to the untagged Rev as assayed by its ability to induce expression of another viral protein Gag (Stauber et al, 1998a).…”

“…The Rev::GFPsg fusion gene was excised from a pBluescript-Rev::GFPsg plasmid (Stauber et al, 1998a) as a BglII/XbaI fragment and subcloned into pUAST P-element vector (Brand and Perrimon, 1993). Together with helper plasmid p 25.7wc⌬2-3, pUAST-Rev::GFPsg plasmids were co-injected into y 1 w 1 embryos and transgenic flies were generated.…”