Abstract
BackgroundThe 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.Methodology/Principal FindingsA 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°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 KV3.4 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/SignificanceThe 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.
Highlights
The ability to ablate the function of a specific protein is a powerful tool for investigating protein function
The genetically encoded substrate consisted of a donor, Cerulean [15], and an acceptor, Venus [16], fluorescent protein (FP) joined by a linker containing a tobacco etch virus NIa protease (TEVp) recognition sequence (TRS; Fig. 1A)
We have shown that protein function can be altered using a rapamycin-inducible TEVp and a TEVp substrate recognition sequence (TRS) inserted into a protein of interest
Summary
The ability to ablate the function of a specific protein is a powerful tool for investigating protein function. Genetic approaches are used to reduce or eliminate protein expression by targeting the gene coding for the protein of interest (genetic knock-out) or interfering with gene transcription or translation (RNA interference, antisense RNA) These methods target protein synthesis and turnover of endogenous protein must occur before a phenotype is realized. Cleavage of substrate by this fraction of enzyme is evident when the total concentration of sTEVp fragments (Fig. 2) or the apparent enzyme concentration is increased (cis constructs and membrane-targeted constructs; Fig. 4 and 5). In these examples, for a given amount of TEVp fragments, the proportion of the TEVp fragments that exist as functional proteases is not predicted to change.
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