Cell-free expression of a GFP fusion protein allows quantitation in vitro and in vivo

The green fluorescent protein (GFP) of Aequorea victoria is frequently fused to other proteins to serve as a reporter for gene expression or the localization of proteins in vivo. We report that when a GFP fusion protein is translated in vitro under standard conditions, the GFP portion folds efficiently and becomes fluorescent. This provides a convenient method for monitoring in vitro translation efficiency of a fusion protein, and to screen for improved mutants of GFP. In addition, quantitation of the translation product combined with fluorescence microscopy of the product immunoprecipitated onto beads allows the determination of the density of the fusion protein in microscopic images.A fusion of the tobacco mosaic virus 30 kDa movement protein [1xInteraction of tobamovirus movement proteins with the plant cytoskeleton. Heinlein, M, Epel, BL, Padgett, HS, and Beachy, RN. Science. 1995; 270: 1983–1985Crossref | PubMedSee all References[1] to the amino terminus of the S65T mutant of GFP [2xImproved green fluorescence. Heim, R, Cubitt, AB, and Tsien, RY. Nature. 1995; 373: 663–664Crossref | PubMedSee all References[2] was translated using a standard rabbit reticulocyte lysate system incorporating 35S-labeled methionine of known specific radioactivity. The products were electrophoresed using SDS and polyacrylamide gel electrophoresis (SDS–PAGE) followed by autoradiography (Figure 1aFigure 1a, lane 1). The major product exhibited the expected mobility; minor products of greater mobility are presumed to arise from internal initiation or premature termination. Scintillation counting, combined with knowledge of the number of methionine residues per molecule of the fusion protein, showed that each microliter of the translation reaction contained 3.8 × 10−15 moles of fusion protein.Figure 1(a) Autoradiogram of translation products separated by SDS–PAGE. The cDNA encoding the fusion protein was excised from the pTMVM:Gfus plasmid [1xInteraction of tobamovirus movement proteins with the plant cytoskeleton. Heinlein, M, Epel, BL, Padgett, HS, and Beachy, RN. Science. 1995; 270: 1983–1985Crossref | PubMedSee all References[1] and inserted into pSP64T [6xFunctional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Krieg, PA and Melton, DA. Nucleic Acids Res. 1984; 12: 7057–7070Crossref | PubMed | Scopus (804)See all References[6]. SP6 transcript (3 μg) was added to 60 μl rabbit reticulocyte lysate (Promega) containing [35S]methionine. The control sample had no transcript added. Reactions were allowed to proceed for 2 h at 25°C, and were then kept at 16°C for 4 h. To remove unincorporated [35S]methionine, the samples were diluted with phosphate-buffered saline (PBS) and filtered four times in Millipore ultrafree-MC low binding regenerated cellulose filter units with a nominal molecular weight limit of 30 kDa, which had been prewashed with 1% bovine serum albumin (BSA), 1% sucrose. Lane 1, translation product; lane 2, control sample (no RNA); lane 3, immunoprecipitated translation product; lane 4, immunoprecipitated control sample (no RNA). Each lane contains the equivalent of a 0.25 μl sample of the original reaction. Markers (in kDa) on the left. (b) Fluorescence emission spectra of fusion protein translated in vitro, diluted to various extents in PBS, and of a control (no RNA) sample. The excitation wavelength was 475 nm. A Hitachi F-2000 fluorescence spectrophotometer was used, with a 10 nm bandpass for both the excitation and emission monochrometers. Fluorescence was stable for at least 3 days at 4°C. (c) White light and fluorescence micrographs of protein G–sepharose beads used to immunoprecipitate varying amounts of fusion protein translated in vitro. Each immunoprecipitation reaction contained 1 μl protein G–sepharose 4 fast-flow bead slurry (Pharmacia), containing about 630 beads, and 0.5 μl anti-GFP antibody (Clontech), in 500 μl PBS containing 0.2% Triton X-100 (Sigma). The reactions were shaken at 4°C for 16 h, and the beads were washed once with PBS containing 0.2% Triton X-100. Scintillation counting showed that the efficiency of the immunoprecipitation was about 60% regardless of the amount of translation reaction added to the beads. The beads were examined with a Nikon Optiphot2-UD microscope equipped with a B-2A FITC filter set (450–490 excitation filter, 505 dichroic mirror, 520 barrier filter) using an objective with a numerical aperture of 0.50, and were photographed on Kodak Ektachrome P1600 film. Beads used to immunoprecipitate a control reaction (no RNA) showed no fluorescence (not shown).View Large Image | View Hi-Res Image | Download PowerPoint SlideThe GFP part of the fusion protein folded properly, as shown by its fluorescence emission spectrum (Figure 1bFigure 1b). A clear signal was readily detected in as little as 2 μl of the translation reaction diluted 100-fold, corresponding to a fusion protein concentration of approximately 4 × 10−11 M.The fusion protein was immunoprecipitated onto protein G–sepharose beads using an anti-GFP antibody. Only the major translation product and a minor product, presumed to arise from internal initiation 20 aminoacids downstream from the amino terminus, were recognized by the antibody (Figure 1aFigure 1a, lane 3).Fluorescence from the beads was easily detectable by fluorescence photomicroscopy when the amount of translation reaction added to an immunoprecipitation corresponded to as little as 1 nl per bead (Figure 1cFigure 1c). This is equivalent to about 2 × 106 protein molecules per bead, giving a surface density of about 80 molecules per μm2. Even a density as low as about 8 molecules per μm2 could be seen in the microscope (data not shown). The true density of fluorescent molecules may be even lower than indicated, as the fraction of synthesized molecules that adopted a fluorescent conformation is not known. Each bead is somewhat larger than a eukaryotic cell (90 μm in diameter), suggesting that a similarly small number of GFP-fusion molecules could be detected in a living cell, particularly if the protein were localized; autofluorescence from a cell, however, might place a lower limit on this number [3xQuantitative imaging of green fluorescent protein in cultured cells: comparison of microscopic techniques, use in fusion proteins and detection limits. Niswender, KD, Blackman, SM, Rohde, L, Magnuson, MA, and Piston, DW. J Microsc. 1995; 180: 109–116Crossref | PubMedSee all References[3].Cell-free expression of GFP seems ideal for the rapid development of new GFP mutants with brighter fluorescence or shifted spectra, based on the recently determined crystal structure [[4]xCrystal structure of the Aequorea victoria green fluorescent protein. Ormo, M, Cubitt, AB, Kallio, K, Gross, LA, Tsien, RY, and Remington, SJ. Science. 1996; 273: 1392–1395Crossref | PubMedSee all References, [5]xThe molecular structure of green fluorescent protein. Yang, F, Moss, LG, and Phillips, GN Jr. Nature Biotechnol. 1996; 14: 1246–1251CrossrefSee all References]. Large numbers of mutants could be screened rapidly by using cell-free coupled transcription–translation of cDNA clones in microtiter plates. In addition, the fusion of GFP to other proteins provides a simple nonradioactive method of measuring translation efficiency, which could expedite the optimization of translation of any protein of interest. Furthermore, if radiolabeled amino acids are incorporated, fluorescence intensity can be correlated with protein quantity, allowing the determination of the density of GFP-fusion protein molecules in a microscopic image of a living cell, a task which cannot easily be accomplished by analysis in vivo.