Engineering a temperature sensitive tobacco etch virus protease

: Since tobacco etch virus protease (TEVp) has a high specificity and efficiency in cleaving its target substrates, many groups have attempted to engineer conditional control of its activity. Temperature induction is widely used for modulating gene function because it has fast temporal response, good penetrability and applicability to many model organisms. Here, we engineered a temperature sensitive TEVp (tsTEVp) by using N-terminal truncations to TEVp that achieved efficient proteolysis on a timescale of 4 h after 30°C induction, while remaining relatively inactive at 37°C. As demonstration, tsTEVp was used to generate temperature-induced biological responses for protein translocation, protein degradation and Ca2+-mediated cellular blebbing. Lastly, tsTEVp and their engineered target substrates could find applications in engineered synthetic biological systems.