2INSERM U563, Département Innovation Thérapeutique et Oncologie Moléculaire, Institut Claudius Régaud, Université Paul Sabatier, Toulouse, France
3Université Paul Sabatier, IPBS, Toulouse, France
4Bioscience Division, MS-M888, Los Alamos National Laboratory, Los Alamos, NM, USA
Although epitope tags are useful to detect intracellular proteins and follow their localization with antibodies, background and nonspecific staining often remain problematic. We describe a simple assay based on the split GFP complementation system. Proteins tagged with the 15–amino acid GFP 11 fragment are detected with a solution of the recombinant nonfluorescent complementary GFP 1-10 fragment to reconstitute a fluorescent GFP. In contrast to antibody-based staining methods, this one-step assay presents high specificity and very low background of fluorescence, thus conferring higher signal-to-noise ratios. We demonstrate that this new application of the split GFP tagging system facilitates detection of proteins displaying various subcellular localizations using flow cytometry and microscopy analysis.
Understanding protein dynamics in living cells requires sensitive and quantitative tools for measuring spatiotemporal modifications of protein expression and/or localization. Fusion to green fluorescent protein (GFP) or its derivatives enables direct visualization of intracellular proteins without the need for secondary reagents or treatment of the cell (1). However, the large size of GFP may alter protein localization and behavior (2), and the permanent fluorescence of the reporter can be a hindrance to detect other markers. To detect native intracellular proteins, monoclonal or polyclonal antibodies are sometimes available, but they need to recognize an epitope conserved after fixation. Alternatively, various epitope tags (myc, His, HA, Flag) have been developed to specifically detect or isolate proteins in cells (3). Detection of such tags either involves specific monoclonal antibodies directly linked to a fluorochrome or successive steps of unlabeled antibody and fluorochrome-conjugated secondary reagents (4). Such protocols are often time-consuming, as they require several washing steps to remove all unbound reagents between binding reactions. Moreover, low signal-to-noise ratios are often observed, due to the presence of nonspecifically bound antibodies, especially for polyclonal antibodies, or due to endogenous expression of the epitope in the parent cell (5).
As a possible alternative, we have investigated whether we could use the split GFP tagging system for the intracellular detection of proteins (6), which is based on the auto-assembly capacity of two nonfluorescent portions of GFP—GFP 1-10 and GFP 11—to restore a fully fluorescent GFP. The GFP 11 tag, which is only 15 amino acids long, is fused to the N or C terminus of the coding sequence of the protein of interest and can then be expressed in eukaryotic cells. The GFP 1-10 detector fragment is produced separately in Escherichia coli and purified from inclusion bodies as previously described (7). After fixation and permeabilization of cells expressing the GFP 11–tagged protein, the refolded GFP 1-10 protein is added in trans, allowing the two split GFP fragments to associate spontaneously and restore the GFP fluorescence (Figure 1A). Here, we describe the application of the split GFP protein complementation assay for detecting GFP 11–tagged proteins in mammalian cells relative to antibody staining using FACS and microscopy analysis.
Materials and methods Plasmids
pcDNA 3.1 vector expressing human MeCP2e1-Myc-His was provided by Dr. Berge A. Minassian of the Hospital for Sick Children, Toronto, Ontario, Canada (8). For FK506 binding protein 12 and MeCP2e1, the respective coding sequences were inserted at the N terminus of GFP 11, in a vector derived from pEGFP_N3 (Clontech Laboratories, Saint-Germain-en-Laye, France) (see sequence of the mammalian GFP 11 cassette below). To generate GFP 11–H-Ras, full-length GFP was replaced by the 15–amino acid mammalian GFP 11 peptide (GFP11m) in the pEGFP-H-Ras plasmid kindly provided by J. Lippincot-Schwartz (9).DNA sequence of the GFP11m vector cassette
GFP11m N-terminal fusion. 5′-ATGGGCCGGGACCACATGGTGCTGCACGAGTACGTGAACGCCGCCGGCATCACAGACGGCGGCAGCGGCGGCGGCAGC-3′.
GFP11m C-terminal fusion. 5′-GGCGACGGCGGCAGCGGCGGCGGCAGCCGGGACCACATGGTGCTGCACGAGTACGTGAACGCCGCCGGCATCACATAA-3′.
Recombinant GFP 1-10. Expression of GFP 1-10 detection reagent was performed in E. coli BL21 (DE3). Recombinant protein was purified from inclusion bodies as described previously (7). For each set of assays, 37.5 mg purified inclusion bodies were used to prepare 15 mL GFP 1-10 solution (2.5 mg/mL) in 50 mM Tris, pH 7.4, 0.1 M NaCl, 10% glycerol (TNG).Cell culture
Neuro2A (N2A) cells were maintained in DMEM supplemented with 10% FCS, 100 U/mL penicillin, 100 µg/mL streptomycin, 10 mM HEPES, 0.1 mM nonessential amino acids, and 1 mM sodium pyruvate. Human embryonic kidney (HEK) 293 cells were grown in RPMI-1640 medium supplemented with 10% FCS, 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine.Transfection
Transfections were carried out using JetPEI (Polyplus Transfection, Illkirch, France) following the manufacturer's instructions. Depending on the resistance gene carried by the plasmid vector, stable clones were selected with either 500 µg /mL G418 (Invitrogen, Cergy Pontoise, France) or 125 µg/mL Zeocin (InvivoGen, Toulouse, France) and thereafter maintained at these concentrations. Monoclonal cell lines were obtained by single cell dilution in 96-well plates. Cells used as negative control were untransfected, and were thereby not cultured in the presence of selecting drugs.