One of the problems in modern day biomedical research is turning on/off protein expression.
In order to control in vivo protein levels, many researchers have reverted to genetically tractable organisms such as yeast and worms. In the September issue of Cell, there's a cool paper by Banaszynski et al., who developed reagents so that you can pharmacological manipulate the expression of any protein in the cell.
They take advantage of the FKBP-rapamycin-FRB system, where the addition of a drug (rapamycin) promotes the association of two proteins (FKBP and FRB). Using Yellow fluorescent protein-FKBP chimeras and error-prone PCR they screened cells (each expressing a mutant version of the FKBP chimera) by FACS analysis and isolated mutant versions of the FKBP protein that are inherently unstable but become stable in the presence of a rapamycin derivative (Shld-1). The nice thing about the Shld-1 FKBP interaction is that it is reversible.
Take home: you can fuse mutant FKBP to any protein. Your protein of interest is now stable only in the presence of Shld-1.
Step 1: Transfect cells with a plasmid encoding the fusion protein (FKBP-your favorite protein). Want to see what your protein does to cells over short periods of time? Step 2: Add Shld-1 and your favorite protein can be made and is now stable. Want to get rid of your protein? Step 3: Wash out the Shld-1 and your favorite protein is degraded.
Another neat new reagent.
- Log in to post comments
It will be interesting to see if this works well in vivo in transgenic animals. Maybe we'll make some transgenic flies and test it out. Success will hinge on (1) in vivo toxicity of the small-molecule ligand, (2) off-target effects of the small-molecule ligand, (3) stability of the small-molecule ligand when ingested, (4) ability of the small-molecule ligand to gain access to the cells of interest.
Good luck. If it does work in a whole organism that would be very cool. (I guess it would be the equivalent of worms eating RNAi producing E Coli.) Talking to some of my coworkers about the shld-1/FKBP in a whole organism - we weren't sure whether there were any advantages over the cre-lox system for KO-ing endogenous proteins. Would be great how ever to control an over expressed exogenous protein.
"we weren't sure whether there were any advantages over the cre-lox system for KO-ing endogenous proteins"
Cre-Lox is irreversible. The FKBP method is, in principle, reversible.
Good point.