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Efficiently folding and circularly permuted variants of the Sapphire mutant of GFP.

Zapata-Hommer O, Griesbeck O - BMC Biotechnol. (2003)

Bottom Line: The green fluorescent protein (GFP) has been widely used in cell biology as a marker of gene expression, label of cellular structures, fusion tag or as a crucial constituent of genetically encoded biosensors.However, folding of some of these mutants is still a problem when targeted to certain organelles or fused to other proteins.By directed rational mutagenesis, we have produced a new variant of the Sapphire mutant of GFP with improved folding properties that turns out to be especially beneficial when expressed within organelles or as a fusion tag.

View Article: PubMed Central - HTML - PubMed

Affiliation: Max-Planck-Institut für Neurobiologie Am Klopferspitz 18a 82152 Martinsried, Germany. zapata@neuro.mpg.de

ABSTRACT

Background: The green fluorescent protein (GFP) has been widely used in cell biology as a marker of gene expression, label of cellular structures, fusion tag or as a crucial constituent of genetically encoded biosensors. Mutagenesis of the wildtype gene has yielded a number of improved variants such as EGFP or colour variants suitable for fluorescence resonance energy transfer (FRET). However, folding of some of these mutants is still a problem when targeted to certain organelles or fused to other proteins.

Results: By directed rational mutagenesis, we have produced a new variant of the Sapphire mutant of GFP with improved folding properties that turns out to be especially beneficial when expressed within organelles or as a fusion tag. Its absorption spectrum is pH-stable and the pKa of its emission is 4.9, making it very resistant to pH perturbation inside cells.

Conclusion: "T-Sapphire" and its circular permutations can be used as labels of proteins or cellular structures and as FRET donors in combination with red-fluorescent acceptor proteins such as DsRed, making it possible to completely separate donor and acceptor excitation and emission in intensity-based FRET experiments.

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Related in: MedlinePlus

Long emission wavelength genetic indicator of protease activity Fusion of T-Sapphire to DsRed. The GFPs are linked by a protease cleavage site specific for enterokinase (EK). A shows the excitation and emission spectra of the donor T-Sapphire (green) and DsRed (red) individually. B shows the emission spectrum of the fusion protein before (thick solid line) and at 10 min, 30 min, 1h and 1h 30 min (dotted lines) after digestion with Enterokinase. Digestion by enterokinase completely separates the fluorophores from each other after 1hr 30mins. Excitation of the fusion protein was at 399 nm. Note the complete separation of the donor and acceptor spectra after digestion with enterokinase.
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Figure 3: Long emission wavelength genetic indicator of protease activity Fusion of T-Sapphire to DsRed. The GFPs are linked by a protease cleavage site specific for enterokinase (EK). A shows the excitation and emission spectra of the donor T-Sapphire (green) and DsRed (red) individually. B shows the emission spectrum of the fusion protein before (thick solid line) and at 10 min, 30 min, 1h and 1h 30 min (dotted lines) after digestion with Enterokinase. Digestion by enterokinase completely separates the fluorophores from each other after 1hr 30mins. Excitation of the fusion protein was at 399 nm. Note the complete separation of the donor and acceptor spectra after digestion with enterokinase.

Mentions: In order to test whether T-Sapphire can be used as a donor molecule in FRET-based types of indicators we constructed fusion molecules consisting of T-Sapphire and DsRed, linked by a 25 residue cleavable sequence containing a mammalian enterokinase recognition site specifically proteolyzed by enteropeptidase. Previously, Sapphire had been used once as a donor in genetically encoded calcium indicators [12]. Unfortunately, no spectra of these indicators had been displayed in this study leaving some of the properties of these probes unclear. Purified fusion proteins were examined spectroscopically. Figure 3A for clarity shows the excitation and emission spectra of T-Sapphire and DsRed as single molecules. In Figure 3B the emission spectrum of the T-Sapphire\DsRed fusion molecule is depicted before (solid line) and at various time points after the start of digestion with enteropeptidase (dotted lines). The T-Sapphire/DsRed fusion protein showed detectable FRET as evaluated from the emisson spectrum of the intact protein because excitation of T-Sapphire at 399 nm resulted in substantial red emission (Fig. 3B). Digesting the cleavable linker between the two fluorescent proteins separated the two fluorophores from each other and disrupted FRET. After 1hr 30 min of digestion, no further changes in spectra could be observed indicating that the cleavage had proceeded to completion, as verified by PAGE (data not shown). Interestingly, the DsRed emission was completely abolished after separation of the two fluorophores demonstrating that it is possible to specifically excite the donor and separate the emissions of the donor and acceptor molecules completely using this combination of fluorescent proteins. In an independent experiment, digestion with enterokinase was not found to affect fluorescence of DsRed or T-Sapphire directly. For comparison, identical fusion proteins were constructed using CFP or EGFP as donors (data not shown). With these donors spectra could not be separated completely. Small emission maxima at 583 nm still seen after complete cleavage of CFP-DsRed and EGFP-DsRed are due to cross-excitation of DsRed at the given wavelengths of 432 nm and 488 nm, respectively, which was unavoidable due to the complex excitation spectrum of DsRed.


Efficiently folding and circularly permuted variants of the Sapphire mutant of GFP.

Zapata-Hommer O, Griesbeck O - BMC Biotechnol. (2003)

Long emission wavelength genetic indicator of protease activity Fusion of T-Sapphire to DsRed. The GFPs are linked by a protease cleavage site specific for enterokinase (EK). A shows the excitation and emission spectra of the donor T-Sapphire (green) and DsRed (red) individually. B shows the emission spectrum of the fusion protein before (thick solid line) and at 10 min, 30 min, 1h and 1h 30 min (dotted lines) after digestion with Enterokinase. Digestion by enterokinase completely separates the fluorophores from each other after 1hr 30mins. Excitation of the fusion protein was at 399 nm. Note the complete separation of the donor and acceptor spectra after digestion with enterokinase.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC161811&req=5

Figure 3: Long emission wavelength genetic indicator of protease activity Fusion of T-Sapphire to DsRed. The GFPs are linked by a protease cleavage site specific for enterokinase (EK). A shows the excitation and emission spectra of the donor T-Sapphire (green) and DsRed (red) individually. B shows the emission spectrum of the fusion protein before (thick solid line) and at 10 min, 30 min, 1h and 1h 30 min (dotted lines) after digestion with Enterokinase. Digestion by enterokinase completely separates the fluorophores from each other after 1hr 30mins. Excitation of the fusion protein was at 399 nm. Note the complete separation of the donor and acceptor spectra after digestion with enterokinase.
Mentions: In order to test whether T-Sapphire can be used as a donor molecule in FRET-based types of indicators we constructed fusion molecules consisting of T-Sapphire and DsRed, linked by a 25 residue cleavable sequence containing a mammalian enterokinase recognition site specifically proteolyzed by enteropeptidase. Previously, Sapphire had been used once as a donor in genetically encoded calcium indicators [12]. Unfortunately, no spectra of these indicators had been displayed in this study leaving some of the properties of these probes unclear. Purified fusion proteins were examined spectroscopically. Figure 3A for clarity shows the excitation and emission spectra of T-Sapphire and DsRed as single molecules. In Figure 3B the emission spectrum of the T-Sapphire\DsRed fusion molecule is depicted before (solid line) and at various time points after the start of digestion with enteropeptidase (dotted lines). The T-Sapphire/DsRed fusion protein showed detectable FRET as evaluated from the emisson spectrum of the intact protein because excitation of T-Sapphire at 399 nm resulted in substantial red emission (Fig. 3B). Digesting the cleavable linker between the two fluorescent proteins separated the two fluorophores from each other and disrupted FRET. After 1hr 30 min of digestion, no further changes in spectra could be observed indicating that the cleavage had proceeded to completion, as verified by PAGE (data not shown). Interestingly, the DsRed emission was completely abolished after separation of the two fluorophores demonstrating that it is possible to specifically excite the donor and separate the emissions of the donor and acceptor molecules completely using this combination of fluorescent proteins. In an independent experiment, digestion with enterokinase was not found to affect fluorescence of DsRed or T-Sapphire directly. For comparison, identical fusion proteins were constructed using CFP or EGFP as donors (data not shown). With these donors spectra could not be separated completely. Small emission maxima at 583 nm still seen after complete cleavage of CFP-DsRed and EGFP-DsRed are due to cross-excitation of DsRed at the given wavelengths of 432 nm and 488 nm, respectively, which was unavoidable due to the complex excitation spectrum of DsRed.

Bottom Line: The green fluorescent protein (GFP) has been widely used in cell biology as a marker of gene expression, label of cellular structures, fusion tag or as a crucial constituent of genetically encoded biosensors.However, folding of some of these mutants is still a problem when targeted to certain organelles or fused to other proteins.By directed rational mutagenesis, we have produced a new variant of the Sapphire mutant of GFP with improved folding properties that turns out to be especially beneficial when expressed within organelles or as a fusion tag.

View Article: PubMed Central - HTML - PubMed

Affiliation: Max-Planck-Institut für Neurobiologie Am Klopferspitz 18a 82152 Martinsried, Germany. zapata@neuro.mpg.de

ABSTRACT

Background: The green fluorescent protein (GFP) has been widely used in cell biology as a marker of gene expression, label of cellular structures, fusion tag or as a crucial constituent of genetically encoded biosensors. Mutagenesis of the wildtype gene has yielded a number of improved variants such as EGFP or colour variants suitable for fluorescence resonance energy transfer (FRET). However, folding of some of these mutants is still a problem when targeted to certain organelles or fused to other proteins.

Results: By directed rational mutagenesis, we have produced a new variant of the Sapphire mutant of GFP with improved folding properties that turns out to be especially beneficial when expressed within organelles or as a fusion tag. Its absorption spectrum is pH-stable and the pKa of its emission is 4.9, making it very resistant to pH perturbation inside cells.

Conclusion: "T-Sapphire" and its circular permutations can be used as labels of proteins or cellular structures and as FRET donors in combination with red-fluorescent acceptor proteins such as DsRed, making it possible to completely separate donor and acceptor excitation and emission in intensity-based FRET experiments.

Show MeSH
Related in: MedlinePlus