A new way to rapidly create functional, fluorescent fusion proteins: random insertion of GFP with an in vitro transposition reaction

Abstract

Background: The jellyfish green fluorescent protein (GFP) can be inserted into the middle of another protein to produce a functional, fluorescent fusion protein. Finding permissive sites for insertion, however, can be difficult. Here we describe a transposon-based approach for rapidly creating libraries of GFP fusion proteins.

Results: We tested our approach on the glutamate receptor subunit, GluR1, and the G protein subunit, alphas. All of the in-frame GFP insertions produced a fluorescent protein, consistent with the idea that GFP will fold and form a fluorophore when inserted into virtually any domain of another protein. Some of the proteins retained their signaling function, and the random nature of the transposition process revealed permissive sites for insertion that would not have been predicted on the basis of structural or functional models of how that protein works.

Conclusion: This technique should greatly speed the discovery of functional fusion proteins, genetically encodable sensors, and optimized fluorescence resonance energy transfer pairs.

Figure 1

Transposition with <EGFP-V> (A) The transposon, <EGFP-V>, is flanked by 19 bp inverted repeats, the MEs. The EGFP coding region is positioned such that when <EGFP-V> inserts between the codons of a target gene, a fusion protein will be produced. <EGFP-V> also carriesKan^r. There is a stop codon in the 5' end of the Kan^r cassette in the same frame as the EGFP coding sequence, so if the transposon lands in an open reading frame, in the correct orientation and frame, a truncated, EGFP-tagged, protein will initially be produced. Removal of theKan^r cassette by Srf I digestion and re-ligation produces a reading frame that extends across the entire transposon. (B) The target plasmid, α_sEE in pcDNA1/Amp, encodes an epitope tagged version of the G protein subunit α_s. (C) Transposed plasmids carry Amp^r and Kan^r. <EGFP-V> insertions within the target gene produce a PCR product when <EGFP-V> is inserted in the correct orientation, and the size of the PCR product reveals which <EGFP-V> insertions are in the coding sequence.

Figure 2

<EGFP-V> Insertions in α_sEE. (A) Location of 35 <EGFP-V> insertions in the α_sEE coding region. The 13 labeled insertions marked above the coding region are in the correct reading frame and encode fluorescent fusion proteins (12 unique insertions, 1 duplication). The insertions are named for the 3 amino acids of α_s duplicated during transposition. The 22 unlabeled insertions (18 unique) marked below the coding region are those in which <EGFP-V> landed out-of-frame with respect to α_sEE. Redundant insertions are indicated by the number of clones recovered at that site (e.g. 2X, 4X). (B) Srf I digestion of the transposed clone, followed by religation, removes theKan^r selection cassette and produces the full-length fusion protein. In the final fusion protein, the EGFP domain is bordered by amino acid linkers encoded by the Tn5 MEs as well as the 9 bp duplication of the target sequence that is generated during the transposition process.

Figure 3

Model of α_s-GFP(92–94). The GFP insertions into α_s can be interpreted in the context of the structures of GFP (PDB file: 1EMA) and α_s-GTPγ S (PDB file: 1AZT). In this image, the structure of GFP [42] is green, while the helical domain of the α_subunit [15] is pink, and the GTPase domain is blue. GTPγ S is yellow. The GFP insertion α_s-GFP(92–94) that produced a functional G protein subunit is illustrated by the short linkers (encoded by the Tn5 MEs) between GFP and α_s (dark blue). The other sites of <EGFP-V> insertion are shown as green spheres. The numbers on the spheres indicate the second of the three duplicated residues that flank the transposon insertions (the numbers are based on the long form of α_s).

Figure 4

Localization of α_s-GFP Fusion Proteins in Living Cells. (A) Membrane localization of tribrid fusion, α_s-GFP(92–94), in HEK 293 cells ~24 hr after co-transfection with β₁ and γ₇. Similar localization patterns were observed when α_s-GFP(18–20), α_s-GFP(N), or α_s-GFP(C) fusions were co-expressed with β₁ and γ₇ (A non-linear representation of the image brightness was used to illustrate both the dimly fluorescent cells in the upper right corner and the very bright ones at the bottom, scale bar = 20 μm). (B) The remaining 10 tribrid fusion proteins were evenly distributed throughout the cytosol (with little fluorescence in the nucleus) as seen here in HEK 293 cells transiently expressing α_s-GFP(362–364), β₁ and γ₇.

Figure 5

Activity and Expression Levels of α_s-GFP Fusion Proteins. (A) HEK 293 cells were transfected with 2 μg/10⁶ cells of the indicated α_s-GFP constructs or vector alone (pcDNA1/Amp) and 0.2 μg/10⁶ cells of plasmid encoding the LH receptor. cAMP accumulation was measured in the presence (dark gray bars) or absence (light gray bars) of hCG, an LH receptor agonist. Cells expressing each of the α_s-GFP fusion proteins exhibited increased basal and stimulated cAMP accumulation relative to cells expressing vector alone, but only the increases in cells expressing α_s-GFP(92–94) were significantly greater (p < .05). Values represent the mean ± S.E. of 5 independent experiments. (B) Immunoblots of the membrane pellets (P) and supernatant (S) fractions from transiently transfected HEK 293 cells. Expression levels of tribrid fusion proteins α_s-GFP(18–20) and α_s-GFP(92–94), but not of amino- and carboxy-terminus fusions, α_s-GFP(N) and α_s-GFP(C), respectively, were decreased in both fractions relative to that of unlabeled α_sEE. Similar results were obtained in an additional experiment.

Figure 6

Insertion Sites and Functional Screening of GluR1-GFP Fusion Proteins (A) A model of GluR1 topology showing the locations of the GFP insertion in 45 fluorescent fusion proteins. In-frame insertions of <TgPT-0> result in a 3 amino acid duplication (green) flanking the insertion site. In-frame insertions of <TcPT-1> generate only a 2 amino acid duplication (cyan) in the target because of the different reading frame. The orange amino acids are overlapping insertion sites of the two transposons (See supplemental diagram for the two reading frames). Multiple clones with identical transpositions are identified as 2x, 3x, etc. The six insertions resulting in functional, fluorescent GluR1-GFP/CFP tribrid fusion proteins are identified by the duplicated target amino acids (e.g. g209–211, c867–868). This figure was adapted from [43]. (B) AMPA receptor-mediated current from GluR1-CFP(867–868). (B1) Large whole-cell current elicited by the rapid sustained application of 5 mM glutamate (bar) in a cell transiently expressing GluR1-CFP(867–868) after reducing desensitization with cyclothiazide (100 μM). (B2) Current elicited by 5 mM glutamate in an outside-out patch pulled from a cell transiently expressing GluR1-CFP(867–868). (B3) Current elicited in the same patch as B3, but in the absence of cyclothiazide. Note the rapid and nearly complete desensitization of the current. (B4) The trace on the left is an expanded time scale of B3, the trace on the right is from an outside-out patch pulled from a cell transiently expressing wild-type GluR1 in the presence of 5 mM glutamate without cyclothiazide.