doi: 10.1002/cbic.201000453.
Protease activation of split green fluorescent protein
Affiliations
- PMID: 20945451
- PMCID: PMC3711563
- DOI: 10.1002/cbic.201000453
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Protease activation of split green fluorescent protein
Chembiochem.
.
No abstract available
Figures
Successive steps of proteolysis and fragment association reconstitute GFP.
Pro-GFP is a latent fluorophore activated by proteolysis. A) Thrombin-catalyzed processing of Pro-GFPLVPRG. Fluorescence traces of Pro-GFPLVPRG over time, show an increase in signal in the presence of thrombin protease (pink) but not in the sample lacking protease (blue). Reactions were carried out in duplicate at 27 °C in cleavage buffer, and were monitored for fluorescence (λex/λem, 485 nm/540 nm) at 5-min intervals. Error bars indicate data range from duplicate samples. B) Direct visualization of long-lived signal from protease-treated Pro-GFP. Fluorescence of Pro-GFPLVPRG samples is shown 24 h and 3 weeks after addition of thrombin (right) or buffer only (left). The background tube (BG) contained thrombin and GFP1-10 only. Tubes were imaged with a Typhoon fluorescence scanner (λex/λem, 488 nm/580 nm). C) & D) Processing of Pro-GFPDEVD by the cysteine protease, caspase-3 C) and processing of Pro-GFPGIFLET by HIV protease D). Reaction conditions are as in (A) except that for C), TCEP was included in the assay mixture, and for D), Pro-GFP11GIFLET proteolysis was carried out in MES buffer (pH 6.1) with EDTA (10 mM). The yellow trace in D) is data from samples that contained Pro-GFPGIFLET, HIV protease, and the tight-binding HIV protease inhibitor, nelfinavir (10 μM).
Site-specific selectivity in Pro-GFP activation. A) Extent of reaction. Change in fluorescence (ΔF=Ffinal-Finitial) was measured after a 7.5-h incubation of each potential Pro-GFP substrate, bearing the indicated cleavage site, with 1.5 units of thrombin. Reaction conditions as in Figure 2. B) Continuous monitoring. GFP fluorescence (λex/λem, 485 nm/540 nm) from the same samples was measured at 5-min intervals..
Pro-GFP detects protease activity in E. coli. A) Activation of Pro-GFPDEVD by co-expressed human caspase-3. E. coli cultures expressing one of each of the indicated Pro-GFP variants in the presence or absence of co-expressed caspase-3 were visualized under white light (top) and UV light (λex/λem at 488 nm/580 nm) (bottom). The two right-most control strains co-express GFP1-10 with or without caspase-3. B) Identification by gel shift of processed Pro-GFPDEVD in strain co-expressing caspase-3. Aliquots of bacterial lysate were resolved by SDS-PAGE (18%) without pre-boiling, and were then imaged with a Typhoon fluorescence scanner (λex/λem at 488 nm/580 nm). C) Quantification of Pro-GFPDEVD activation in soluble bacterial lysates. Average fold increase in GFP fluorescence (λex/λem at 488 nm/540 nm) with caspase-3 coexpression, over four independent experiments. Error bars represent standard deviation. D) Activation of Pro-GFPDEVD by active caspase-3, but not by a catalytically inactive caspase mutant (C163A). Liquid cultures were imaged for fluorescence as in A).
References
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- Breurken M, Lempens EH, Merkx M. Chembiochem. 2010;11:1665. - PubMed
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