Efficiently folding and circularly permuted variants of the Sapphire mutant of GFP - PubMed (original) (raw)

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

Otilia Zapata-Hommer et al. BMC Biotechnol. 2003.

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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Figures

Figure 1

Figure 1

Characterization of T-Sapphire A: Refolding of Sapphire and T-Sapphire from their denatured states prepared by incubation in denaturation buffer (8M urea, 1 mM DTT) at 95°C for 2 min. Recovery of fluorescence (emission at 511 nm and excitation at 399 nm) was initiated by 100fold dilution into renaturation buffer (35 mM Kcl, 2 mM MgCl2, 1 mM DTT, 50 mM Tris pH 7.5). B: pH-titration of fluorescence of T-Sapphire. Excitation was at 399 nm and emission at 511 nm. D: Absorbance of equal amounts of T-Sapphire protein at pH 4, 5, 6 and 7. No significant differences were detected.

Figure 2

Figure 2

Targeted expression in HEK293 cells A: Schematic representation of the constructs used for targeting. CR is the signal peptide of calreticulin, KDEL is the ER retention motive. B: Fluorescence emission spectrum of dissociated HEK293 cells transfected for 2 days either with ER-Sapphire or ER-T-Sapphire. Excitation was at 399 nm. C: Individual 293 cell co-expressing ER-T-Sapphire and Yellow Cameleon 3ER. Excitation was either at 400 nm to specifically excite T-Sapphire or 488 to excite the YFP component of YC3ER. Note the identical fluorescence pattern. For comparison, T-Sapphire expressed in the cytosol fills the whole cell including the nucleus (bottom). The emission filter was 535/25. Scale bar 10 μM.

Figure 3

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.

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