Destabilizing domain derived from human FKBP12

Destabilizing domain (DD) derived from human FKBP12, carrying F36V and L106P. Fused to a protein of interest, it targets the whole fusion for degradation, and the cell-permeable ligand Shield-1 shields the fusion from degradation, so the protein level follows the ligand.

Length: 321 bp (107 aa)

Uses: degron

Cleavable: No

Origin: Human FKBP12 F36V with the L106P destabilizing mutation

Characteristics

Encodes a 107-amino-acid protein of 321 nucleotides, beginning GVQVETIS and ending DVELLKPE, with Val36 and Pro106. In NIH3T3 cells L106P is the most destabilizing of five mutants tested. A YFP fusion shows 1-2% of the fluorescence seen with Shield-1, and is negligible within 4 hr of Shield-1 withdrawal. Rescue has an EC50 of about 100 nM Shield-1. Degradation is proteasome-dependent, at least in part.

Applications: Ligand-dependent control of protein level by fusion at either terminus. L106P-YFP fusions show Shield-1-dependent stability in NIH3T3, HEK 293T, HeLa and COS-1 cells. Across DD variants, 14 proteins tested in NIH3T3 cells, including kinases, small GTPases, cell cycle regulators, CREB and the transmembrane protein CD8 alpha, showed ligand-dependent stability.

Limitations: The fusion carries an added 107-residue domain. Protein is lost within hours once Shield-1 is withdrawn, so the ligand must be present to maintain the level. For L106P, residual YFP signal without ligand is 1-2% for N-terminal and about 10% for C-terminal fusions in NIH3T3 cells, and 5-26% in transient transfection. The protein of interest must stay functional as a fusion.

Sequence

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References

  1. Banaszynski et al. (2006). A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules. Cell - Banaszynski 2006 FKBP Destabilizing Domain