Coding

Part:BBa_K1965010

Designed by: Nik Franko   Group: iGEM16_Slovenia   (2016-10-17)

Introduction

Luciferase reporter of the proteolytic activity can be designed either to lead to the decrease of its activity due to proteolysis or to generate the activity by cleavage. Cleavable luciferase assay is expected to be relatively insensitive as it can only detect if a large fraction of the luciferase has been degraded, typically more than 20%, while an assay that leads to the activation of the luciferase might be able to detect much smaller fraction of the proteolytic cleavage.

Into the loop of the firefly luciferase (fLuc) we inserted amino acid sequence that is targeted by proteases. The substrate sequence thus divided the fLuc into two fragments (nLuc and cLuc), with a protease cleavage site between them ( 1 A and B). The insertion site for the substrate sequence was based on the previously described split luciferase system [1], where we expected that this site would also be permissible to short linker insertion without significantly altering luciferase activity. Upon addition of an appropriate protease, the reporter would be cleaved at the substrate site and the two fragments would dissociate and in turn decrease the fLuc activity. In this system higher protease activity corresponds to a lower luciferase activity. The reporters were additionally equipped with a protein tag at the N-terminal and C-terminal end in order to allow immunostaining to detect protein cleavage by the western blot ( 1 ).

Detection of a substrate cleavage by CRY2PHR/CIBN split protease induced by light.(A) Schematic representation of CRY2PHR/CIBN light-inducible system with split protease and substrate with protease target sequence. Activity of (B) TEV, (C) PPV and (D) TEV:E proteases were analyzed by Western blot analysis. 24 hours after transfection of cells with plasmids expressing split proteases and protease substrate, the formation of active protease was induced by light. After the indicated time periods cells were immediately lysed and formation of cleaved products were analysed by Western blot using primary antibodies against AU1 tag. Excluding the non-specific upper band, uncleaved samples show only one band, while the cleaved ones show two bands.

Characterization

For the testing of the activity and orthogonality of different TEV protease variants we used a cleavable firefly luciferase (fLuc) reporter with an appropriate cleavage sequence inserted in a permissible site. We observed a significant decrease in the fLuc activity upon coexpression of the reporters with their corresponding proteases, whereas the coexpression of reporters with an orthogonal protease resulted in a much lower decrease of fLuc activity ( 2 ). These results were additionally confirmed by results from western blot where the cleaved luciferase was detected only in cells cotransfected with a reporter and its corresponding protease but not with other reporter-protease combinations ( 3 ).

Activity and orthogonality of TEVp variants.
HEK293T cells were transfected with the indicated fLuc:TEVs and TEVp variant constructs. Luciferase activity was measured 24h after transfection. The results are presented as normalized firefly luciferase activity (RLU).
Orthogonality and activity of TEVpE and TEVpH.
HEK293T cells were transfected with 2000ng of the indicated protease and 500ng of the indicated reporter. Cells were lysed and analyzed by western blotting against the AU1 tag. The cleaved reporter (55 kDa) was detected only in the presence of the corresponding TEVp variant.

References

[1]Shekhawat, S. S., Porter, J. R., Sriprasad, A., & Ghosh, I. (2009). An Autoinhibited Coiled-Coil Design Strategy for Split-Protein Protease Sensors. Journal of the American Chemical Society, 131(42), 15284–15290. doi:10.1021/ja9050857

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