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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

Ligand-dependent stability of new DDs screened at 25°C.NIH 3T3 cells were stably transduced with the indicated ecDHFR mutants fused to YFP and treated with vehicle (–) or 10 µM trimethoprim (+) for 24 hours at 25°C. (37°C DDs are the ones engineered at 37°C; same mutants as Y-D and D-Y in Figure 2).
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pone-0072393-g003: Ligand-dependent stability of new DDs screened at 25°C.NIH 3T3 cells were stably transduced with the indicated ecDHFR mutants fused to YFP and treated with vehicle (–) or 10 µM trimethoprim (+) for 24 hours at 25°C. (37°C DDs are the ones engineered at 37°C; same mutants as Y-D and D-Y in Figure 2).

Mentions: Populations of NIH 3T3 cells encoding these two DD libraries underwent three rounds of fluorescence-activated cell sorting (FACS) screening. We first selected cells that highly express YFP following 24 hours of treatment with 10 µM trimethoprim at 37°C (The treatment was done at 37°C, not 25°C, for this first round because we were concerned that we would purge out too much portion of the library from the start). Next, we selected cells with low YFP levels in the absence of trimethoprim. For this second round of screening, the cells were cultured at 37°C for the most time, but 24 hours prior to FACS, they were shifted to 25°C. For the third and final round of screening, we selected cells expressing high levels of YFP following 24 hours of treatment with 10 µM trimethoprim. Specifically, the cells were cultured at 37°C prior to the addition of trimethoprim, and the cells were shifted to 25°C during the 24-hour ligand dosing period. After three rounds of FACS, genomic DNA was extracted, and genome-integrated DDs were amplified by PCR. For both the N-terminal and C-terminal libraries, 25 clones were chosen and sequenced. The candidate DDs were stably transduced back into NIH 3T3 cells, and YFP expression levels were measured in the absence and presence of 10 µM trimethoprim. Figure 3 shows clones from both libraries that display ligand-dependent stability (sequence information shown in Table 1). In the absence of trimethoprim (mock-treated with DMSO), the expression levels of YFP-fused DDs drop 25–80 fold.


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)

Ligand-dependent stability of new DDs screened at 25°C.NIH 3T3 cells were stably transduced with the indicated ecDHFR mutants fused to YFP and treated with vehicle (–) or 10 µM trimethoprim (+) for 24 hours at 25°C. (37°C DDs are the ones engineered at 37°C; same mutants as Y-D and D-Y in Figure 2).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0072393-g003: Ligand-dependent stability of new DDs screened at 25°C.NIH 3T3 cells were stably transduced with the indicated ecDHFR mutants fused to YFP and treated with vehicle (–) or 10 µM trimethoprim (+) for 24 hours at 25°C. (37°C DDs are the ones engineered at 37°C; same mutants as Y-D and D-Y in Figure 2).
Mentions: Populations of NIH 3T3 cells encoding these two DD libraries underwent three rounds of fluorescence-activated cell sorting (FACS) screening. We first selected cells that highly express YFP following 24 hours of treatment with 10 µM trimethoprim at 37°C (The treatment was done at 37°C, not 25°C, for this first round because we were concerned that we would purge out too much portion of the library from the start). Next, we selected cells with low YFP levels in the absence of trimethoprim. For this second round of screening, the cells were cultured at 37°C for the most time, but 24 hours prior to FACS, they were shifted to 25°C. For the third and final round of screening, we selected cells expressing high levels of YFP following 24 hours of treatment with 10 µM trimethoprim. Specifically, the cells were cultured at 37°C prior to the addition of trimethoprim, and the cells were shifted to 25°C during the 24-hour ligand dosing period. After three rounds of FACS, genomic DNA was extracted, and genome-integrated DDs were amplified by PCR. For both the N-terminal and C-terminal libraries, 25 clones were chosen and sequenced. The candidate DDs were stably transduced back into NIH 3T3 cells, and YFP expression levels were measured in the absence and presence of 10 µM trimethoprim. Figure 3 shows clones from both libraries that display ligand-dependent stability (sequence information shown in Table 1). In the absence of trimethoprim (mock-treated with DMSO), the expression levels of YFP-fused DDs drop 25–80 fold.

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