Rapid modification of proteins using a rapamycin-inducible tobacco etch virus protease system.
BACKGROUND
The ability to disrupt the function of a specific protein on a rapid time scale provides a powerful tool for biomedical research. Specific proteases provide a potential method to selectively cleave a chosen protein, but rapid control of protease activity is difficult.


RESULTS
A heterologous expression system for rapid target-directed proteolysis in mammalian cells was developed. The system consists of an inducible NIa protease from the tobacco etch virus (TEVp) and a chosen protein into which a TEVp substrate recognition sequence (TRS) has been inserted. Inducible activity was conferred to the TEVp using rapamycin-controlled TEVp fragment complementation. TEVp activity was assayed using a FRET-based reporter construct. TEVp expression was well tolerated by mammalian cells and complete cleavage of the substrate was possible. Cleavage at 37 degrees C proceeded exponentially with a time constant of approximately 150 minutes. Attempts to improve cleavage efficiency were hampered by substantial background activity, which was attributed to inherent affinity between the TEVp fragments. A second TEVp assay, based on changes in inactivation of a modified K(V)3.4 channel, showed that functional properties of a channel can be using altered using an inducible TEVp system. Similar levels of background activity and variability were observed in both electrophysiological and FRET assays.


CONCLUSIONS
The results suggested that an optimum level of TEVp expression leading to sufficient inducible activity (with minimal background activity) exists but the variability in expression levels between cells makes the present system rather impractical for single cell experiments. The system is likely to be more suitable for experiments in which the cell-to-cell variability is less of an issue; for example, in experiments involving large populations of cells.
Williams
Damian J
DJ
Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD, USA.
Puhl
Henry L
HL
Ikeda
Stephen R
SR
D002460:Cell Line; D004594:Electrophysiology / Q000379:methods; D010450:Endopeptidases / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005821:Genetic Techniques; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D017800:Potyvirus / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D051670:Shaw Potassium Channels / Q000737:chemistry; D020123:Sirolimus / Q000737:chemistry*; D013696:Temperature; D013997:Time Factors
C492015:KCNC4 protein, human; D011506:Proteins; D051670:Shaw Potassium Channels; D010450:Endopeptidases; C099488:TEV protease; D020123:Sirolimus