GFP fusion proteins

FIGURE 1.2 In situ localization of the OCP-green fluorescence protein (GFP) fusion protein Immunogold labeling of a thin section of OCP-GFP transformed Synechocystis PCC6803 OCP-GFP cells were labeled with a polyclonal antibody against the GFP coupled to 10 nm gold particles. Bar = 0.5 pm. 

Kahn TW, Beachy RN, Falk MM (1997) Cell-free expression of a GFP fusion protein allows quantitation in vitro and in vivo. Curr Biol 7 R207-R208... 

Vermeer, J. E., Van Munster, E. B., Vischer, N. O. and Gadella, T. W., Jr. (2004). Probing plasma membrane microdomains in cowpea protoplasts using lipidated GFP-fusion proteins and multimode FRET microscopy. J. Microsc. 214, 190-200. 

Alternatively, GFP can be visualized using rabbit polyclonal antibody raised against GFP purified directly from A. victoria. This anti-GFP antibody facilitates the detection of native GFP, recombinant GFP, and GFP-fusion proteins both by immunofluorescence and brightfield microscopy, as well as by western blot analysis and immunoprecipitation. Direct anti-GFP conjugates made from a complete serum or from an IgG fraction are available from Invitrogen (http //www.invitrogen.com/ site/us/en/home.html). Additional options for your research offered by Invitrogen include two mouse monoclonal antibodies and a chicken IgY fraction. 

Monoclonal) antibodies Other characteristics of late endosomes are unique lipids or proteins associated with the different compartments. These can be used as a target for (monoclonal) antibodies. Often, also the recombinant GFP-fusion proteins are available (such as GFP-Rab5 and GFP-RhoB). 

Brock R, Hamelers IHL, Jovin TM. Comparison of fixation protocols for adherent cultured cells applied to a GFP fusion protein of the epidermal growth factor receptor. Cytometry 1999 35 353-362. 

A recent study in Drosophila cells used H3-GFP fusion proteins to compare the deposition patterns of H3.3 and a replication dependent H3 [104]. The deposition of the replication dependent H3-GFP occurred at sites that co-localized with BrdU... 

Kanda, T., Sullivan, K.F., and Wahl, G.M. (1998) Histone-GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells. Curr. Biol. 26 8(7), 377-385. 

Here we describe the construction and use of adenoviruses to express lucif-erase- or GFP-fusion proteins for high-throughput cell-based screens. We constructed adenoviruses that express a cyclin Bl-luciferase reporter protein or unmodified luciferase. This enabled us to screen for small molecules that specifically up-regulated levels of the cyclin B 1-luciferase fusion protein without affecting luciferase. A similar approach was used to construct adenoviruses that express cyclin A-GFP or cyclin Bl-GFP fusion proteins. These viruses were used in an imaging-based screen to identify small molecules that specifically upregulated these proteins or altered their subcellular localization. 

For cells expressing GFP fusion protein, Hoechst 33342 or Draq5 nuclear stain is added to the fixative to ready the cells for image acquisition. For immunofluorescence staining, the cells are incubated with PBS-TB (PBS with 0.2% Triton X-100, 0.1% bovine serum albumin [BSA]) for 10 min to permeabilize the cell membranes. Primary antibody is then added at 0.5 to 5 pg/mL in PBS-TB and incubated at room temperature for 1 hr or at 4°C overnight. The wells are then washed two times with PBS-TB. Fluorescently labeled secondary antibody is added at 2 pg/mL together with 10 pg/mL Hoechst 33342 nuclear stain in PBS-TB and incubated at room temperature for 1 to 2 hr. The cells are then washed with PBS-TB and once with PBS before image acquisition. 

Two recent reports revealed additional layers of complexity regarding the mechanisms involved in the distinct durations of action associated with the different toxin serotypes. Femandez-Salas et al. (2004) investigated the subcellular localization of BoNT/A, /B, and /E LC-GFP fusion proteins following overexpression in several different mammalian cell lines. The LC/A fusion protein was shown to localize within discrete plasma membrane... 

Recently, a photoactivatable variant from Aequoria victoria green fluorescent protein (pa-GFP) was reported (Patterson and Lippincott-Schwartz 2002), yielding an increase in fluorescence emission intensity (at k 520 nm) by a factor of 100 when excited at k 488 nm after spectral activation at A. 408 nm. This phenomenon is due to an internal photoconversion process in the protein and allows spectral photoactivation of this protein in a very local way such as in the nucleus of a living cell (Post et al. 2005). In tobacco BY-2 protoplasts, we transiently co-expressed pa-GFP or pa-GFP fusion proteins and red-fluorescent protein (DsRed)-tagged prenylated Rab acceptor 1 (Pral At2g38360), a membrane protein that localizes in speckles around the nuclear envelope. The DsRed transfection allows proper cell identification and visualization before activation (via Pral -DsRed fluorescence). After pa-GFP... 

Compared with membrane trafficking, which occurs with characteristic rates of 3% per minute for the case of VSVG movement from the endoplasmic reticulum to the Golgi complex (22), the cycling of proteins on and off membranes can occur over very rapidly. In our own work (30), we have observed in pho-tobleaching studies of GFP fusion proteins that a mutant form of HRas that lacks both pahnitoylation sites is able to constitu-tively cycle on and off membranes of the Golgi complex with halftimes of 5 s (Fig. 2). 

This still leaves the question of whether the native receptors adopt this distribution. We took two separate approaches to this. First, we carried out the transfection with the a1B-AR-GFP fusion protein under its own promoter (20). This proved negative in vascular smooth muscle, presumably because the native expression level is low, but positive expression was found in adventitial cells. This not only showed that the transfection/promoter system was effective when the native expression level was high, but also suggested that a1B-AR expression... 

This section indicates that is possible to transfect cells with GFP fusion proteins of the AR families. The co-localization of these proteins with fluorescent ligands provides a link that allows comparison with native cell systems that do not possess such useful tags but that bind the fluorescent ligands. However, an additional utility is provided by the ability to label receptors in native systems with GFP, which provides the opportunity for receptor translocation studies in native cells. This has been accomplished on two levels. First, by in vitro transfection of native tissues or cells (see Fig. 1C) and second by creating transgenic mice harboring the GFP-AR constructs (see Chapter 7). 

Doherty AJ, Collingridge GL, Henley JM (1997) GFP fusion proteins and AMPA receptor trafficking. Biochem... 

Fig. 5.6 Fluorescence microscopy image of BHK cells expressing a human NPY-Y2 re-ceptor-GFP fusion protein.

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