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

Sheridan DL, Berlot CH, Robert A, Inglis FM, Jakobsdottir KB, Howe JR, Hughes TE - BMC Neurosci (2002)

Bottom Line: Here we describe a transposon-based approach for rapidly creating libraries of GFP fusion proteins.We tested our approach on the glutamate receptor subunit, GluR1, and the G protein subunit, alphas.This technique should greatly speed the discovery of functional fusion proteins, genetically encodable sensors, and optimized fluorescence resonance energy transfer pairs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cellular and Molecular Physiology, Yale University Medical School, 330 Cedar St, New Haven, CT 06520, USA. Douglas.Sheridan@yale.edu

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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC117241&req=5

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.

Mentions: The glutamate receptor subunit GluR1 [19] was used to test the new transposons and the visual screening process. Independent transpositions of the GluR1 plasmid were performed with the EGFP and ECFP transposons (<TgPT-0> and <TcPT-1>, respectively). In 288 co-transfections, there were 20 wells with EGFP fluorescence, 21 wells with ECFP fluorescence, and 2 wells with both EGFP and ECFP fluorescence. Sequencing revealed 35 unique insertions (17 <TgPT-0> and 18 <TcPT-1>) and 10 repetitive insertions (figure 6A). The recovery of 45 fluorescent clones from 576 colonies (7.8%) agrees with the predicted frequency of transpositions resulting in GluR1-EGFP/ECFP fusions (7.7%), which is consistent with the interpretation that all in-frame insertions produce a fluorescent protein. Clones representing unique fluorescent fusion proteins were digested with Srf I to remove the Kanr selection cassette and re-ligated to generate full-length GluR1-EGFP/ECFP fusions. These fusion proteins were screened, in transiently transfected HEK 293 cells, for glutamate-gated ion channel function. Of the 29 unique tribrid fusion constructs tested, all produce detectable fluorescence and 6 were functional (figure 6B).


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

Sheridan DL, Berlot CH, Robert A, Inglis FM, Jakobsdottir KB, Howe JR, Hughes TE - BMC Neurosci (2002)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC117241&req=5

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.
Mentions: The glutamate receptor subunit GluR1 [19] was used to test the new transposons and the visual screening process. Independent transpositions of the GluR1 plasmid were performed with the EGFP and ECFP transposons (<TgPT-0> and <TcPT-1>, respectively). In 288 co-transfections, there were 20 wells with EGFP fluorescence, 21 wells with ECFP fluorescence, and 2 wells with both EGFP and ECFP fluorescence. Sequencing revealed 35 unique insertions (17 <TgPT-0> and 18 <TcPT-1>) and 10 repetitive insertions (figure 6A). The recovery of 45 fluorescent clones from 576 colonies (7.8%) agrees with the predicted frequency of transpositions resulting in GluR1-EGFP/ECFP fusions (7.7%), which is consistent with the interpretation that all in-frame insertions produce a fluorescent protein. Clones representing unique fluorescent fusion proteins were digested with Srf I to remove the Kanr selection cassette and re-ligated to generate full-length GluR1-EGFP/ECFP fusions. These fusion proteins were screened, in transiently transfected HEK 293 cells, for glutamate-gated ion channel function. Of the 29 unique tribrid fusion constructs tested, all produce detectable fluorescence and 6 were functional (figure 6B).

Bottom Line: Here we describe a transposon-based approach for rapidly creating libraries of GFP fusion proteins.We tested our approach on the glutamate receptor subunit, GluR1, and the G protein subunit, alphas.This technique should greatly speed the discovery of functional fusion proteins, genetically encodable sensors, and optimized fluorescence resonance energy transfer pairs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cellular and Molecular Physiology, Yale University Medical School, 330 Cedar St, New Haven, CT 06520, USA. Douglas.Sheridan@yale.edu

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.

Show MeSH
Related in: MedlinePlus