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Rapid and tunable control of protein stability in Caenorhabditis elegans using a small molecule.

Cho U, Zimmerman SM, Chen LC, Owen E, Kim JV, Kim SK, Wandless TJ - PLoS ONE (2013)

Bottom Line: To broaden the scope of this technology, we have engineered new destabilizing domains that perform well at temperatures of 20-25°C.We further show that these new destabilizing domains can be used to regulate protein concentrations in C. elegans.These data reinforce that DD can function in virtually any organism and temperature.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Systems Biology, Stanford University, Stanford, California, USA.

ABSTRACT
Destabilizing domains are conditionally unstable protein domains that can be fused to a protein of interest resulting in degradation of the fusion protein in the absence of stabilizing ligand. These engineered protein domains enable rapid, reversible and dose-dependent control of protein expression levels in cultured cells and in vivo. To broaden the scope of this technology, we have engineered new destabilizing domains that perform well at temperatures of 20-25°C. This raises the possibility that our technology could be adapted for use at any temperature. We further show that these new destabilizing domains can be used to regulate protein concentrations in C. elegans. These data reinforce that DD can function in virtually any organism and temperature.

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Related in: MedlinePlus

Temperature dependence of existing DDs.Destabilizing domains derived from either FKBP or ecDHFR were fused to the N-terminus of YFP (F-Y and D-Y) or the C-terminus of YFP (Y-F and Y-D). The indicated fusion proteins were stably expressed in NIH 3T3 cells for 24 h treated with vehicle (–) or stabilizing ligand (+, 2 µM Shield-1 for FKBP-derived DD and 10 µM trimethoprim for ecDHFR-derived DD) at 25°C or 37°C. The expression levels of the fusion proteins were then measured by flow cytometry. (FKBP mutations in F-Y: L106P, Y-F: E31G/R71G/K105E, DHFR mutations in D-Y: R12Y/G67S/Y100I, Y-D: R12H/N18T/A19V/G67S).
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pone-0072393-g002: Temperature dependence of existing DDs.Destabilizing domains derived from either FKBP or ecDHFR were fused to the N-terminus of YFP (F-Y and D-Y) or the C-terminus of YFP (Y-F and Y-D). The indicated fusion proteins were stably expressed in NIH 3T3 cells for 24 h treated with vehicle (–) or stabilizing ligand (+, 2 µM Shield-1 for FKBP-derived DD and 10 µM trimethoprim for ecDHFR-derived DD) at 25°C or 37°C. The expression levels of the fusion proteins were then measured by flow cytometry. (FKBP mutations in F-Y: L106P, Y-F: E31G/R71G/K105E, DHFR mutations in D-Y: R12Y/G67S/Y100I, Y-D: R12H/N18T/A19V/G67S).

Mentions: We tested two of the DD systems by preparing four constructs consisting of the FKBP-derived DD fused to the N- or C-terminus of yellow fluorescent protein (YFP) as well as the ecDHFR-derived DD fused to the N- or C-terminus of YFP. These four constructs were stably introduced into NIH 3T3 cells by retroviral transduction. After 3 days, the stabilizing ligand (Shield-1 for FKBP-derived DDs or trimethoprim for ecDHFR-derived DDs) was added to the cell culture media. Cells were then cultured at 25°C or 37°C for 24 hours before the YFP levels were analyzed by flow cytometry. As expected, the dynamic range was larger at 37°C for all four DD fusions (Figure 2). At 25°C, stabilization of the fusion protein was partial, and degradation in the absence of DD ligand was not fully achieved. Since we are seeking to broaden the scope of the DD technology, we decided to develop a new DD that will provide a large dynamic range when expressed in cells cultured at 25°C. In addition, the development of a low-temperature DD would provide evidence that DDs could be engineered for any desired experimental conditions.


Rapid and tunable control of protein stability in Caenorhabditis elegans using a small molecule.

Cho U, Zimmerman SM, Chen LC, Owen E, Kim JV, Kim SK, Wandless TJ - PLoS ONE (2013)

Temperature dependence of existing DDs.Destabilizing domains derived from either FKBP or ecDHFR were fused to the N-terminus of YFP (F-Y and D-Y) or the C-terminus of YFP (Y-F and Y-D). The indicated fusion proteins were stably expressed in NIH 3T3 cells for 24 h treated with vehicle (–) or stabilizing ligand (+, 2 µM Shield-1 for FKBP-derived DD and 10 µM trimethoprim for ecDHFR-derived DD) at 25°C or 37°C. The expression levels of the fusion proteins were then measured by flow cytometry. (FKBP mutations in F-Y: L106P, Y-F: E31G/R71G/K105E, DHFR mutations in D-Y: R12Y/G67S/Y100I, Y-D: R12H/N18T/A19V/G67S).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3750007&req=5

pone-0072393-g002: Temperature dependence of existing DDs.Destabilizing domains derived from either FKBP or ecDHFR were fused to the N-terminus of YFP (F-Y and D-Y) or the C-terminus of YFP (Y-F and Y-D). The indicated fusion proteins were stably expressed in NIH 3T3 cells for 24 h treated with vehicle (–) or stabilizing ligand (+, 2 µM Shield-1 for FKBP-derived DD and 10 µM trimethoprim for ecDHFR-derived DD) at 25°C or 37°C. The expression levels of the fusion proteins were then measured by flow cytometry. (FKBP mutations in F-Y: L106P, Y-F: E31G/R71G/K105E, DHFR mutations in D-Y: R12Y/G67S/Y100I, Y-D: R12H/N18T/A19V/G67S).
Mentions: We tested two of the DD systems by preparing four constructs consisting of the FKBP-derived DD fused to the N- or C-terminus of yellow fluorescent protein (YFP) as well as the ecDHFR-derived DD fused to the N- or C-terminus of YFP. These four constructs were stably introduced into NIH 3T3 cells by retroviral transduction. After 3 days, the stabilizing ligand (Shield-1 for FKBP-derived DDs or trimethoprim for ecDHFR-derived DDs) was added to the cell culture media. Cells were then cultured at 25°C or 37°C for 24 hours before the YFP levels were analyzed by flow cytometry. As expected, the dynamic range was larger at 37°C for all four DD fusions (Figure 2). At 25°C, stabilization of the fusion protein was partial, and degradation in the absence of DD ligand was not fully achieved. Since we are seeking to broaden the scope of the DD technology, we decided to develop a new DD that will provide a large dynamic range when expressed in cells cultured at 25°C. In addition, the development of a low-temperature DD would provide evidence that DDs could be engineered for any desired experimental conditions.

Bottom Line: To broaden the scope of this technology, we have engineered new destabilizing domains that perform well at temperatures of 20-25°C.We further show that these new destabilizing domains can be used to regulate protein concentrations in C. elegans.These data reinforce that DD can function in virtually any organism and temperature.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Systems Biology, Stanford University, Stanford, California, USA.

ABSTRACT
Destabilizing domains are conditionally unstable protein domains that can be fused to a protein of interest resulting in degradation of the fusion protein in the absence of stabilizing ligand. These engineered protein domains enable rapid, reversible and dose-dependent control of protein expression levels in cultured cells and in vivo. To broaden the scope of this technology, we have engineered new destabilizing domains that perform well at temperatures of 20-25°C. This raises the possibility that our technology could be adapted for use at any temperature. We further show that these new destabilizing domains can be used to regulate protein concentrations in C. elegans. These data reinforce that DD can function in virtually any organism and temperature.

Show MeSH
Related in: MedlinePlus