Part:BBa_K1965044
CIBN:cPPV
Introduction
BBa_K1965044 contains the coding sequence for the truncated version of the CIB1 protein from Arabidopsis thaliana (CIBN), fused to the C-terminal part of split PPV protease. This part works together with BBa_K1965043 (CRY2PHR:nPPV) and is one of the two components of the blue light-inducible dimerization system CRY2/CIB1. For our project, we used the conserved N-terminal photolyase homology region of the CRY2 protein from Arabidopsis thaliana (CRY2PHR; aa 1-498) that mediates light-responsiveness and the truncated version of the CIB1 protein (CIBN; aa 1-170) without the helix-loop-helix region, which mediates DNA binding [1].. Dimerization of CRY2PHR and CIBN can be induced by blue light illumination at approximately 460 nm, after which the interaction occurs on a millisecond timescale and can be reversed within minutes by removing the stimulus. We adapted this system for the reconstitution of split TEV protease to implement blue-light as one of the input signals for protease-based signaling pathway.
Upon induction with light and consequent dimerization of CIBN:cPPV (BBa_K1965044) and CRY2PHR:nPPV (BBa_K1965043), the split PPV protease is reconstituted, resulting protease activity and cleavage of the specific substrate.
Characterization
Activity and orthogonality were tested by cleavage of a cyclic luciferase reporter (BBa_K1965042) with dual luciferase assays . Cleavage of this reporter results in luciferase reconstitution and thus an increase in luminescence (1).
(A)Schematic representation of the CRY2PHR/CIBN light-inducible system with split protease. In the dark, the CRY2PHR and CIBN proteins do not interact (left), while illumination with blue light results in heterodimerization and reconstitution of split protease (right), which in turn cleaves the cyclic luciferase reporter, resulting in increased luciferase activity.. (B) Orthogonality of the split PPV protease. HEK293T cells were transfected with 1:3 ratio of plasmids coding for CRY2PHR:CIBN with split PPVp protease. 24 hours after transfection, cells were illuminated with blue light at 460nm for indicated periods of time. The cells were lysed and bioluminescence was measured with dual luciferase assay. Upon illumination with blue light, each protease cleaves only the cyclic reporter with the correct cleavage site (red bars), while the cyclic reporter with the mismatched cleaage site remains uncleaved (white bars).
Additionally, we tested activity and orthogonality of the light-inducible split protease by cleavage of luciferase with the inserted substrate at the split site after aa 491 with western blotting (Figure 4.9.3.C)The reporter used for the western blot analysis contained the AU1 tag at the N-terminus. Uncleaved luciferase appears as a single band on a western blot (expected size at 65kDa), while partial cleavage of luciferase would in two bands (uncleaved at 65 kDa and cleaved at 55 kDa) and complete cleavage in only the smaller band (Figure 4.9.4A). Our results indicate that substrates carrying specific protease cleavage sites were cleaved after blue light illumination of cells harboring plasmids for expression of CRY2PHR:nPPV and CIBN:cPPV proteins ((2).
(A) Schematic representation of the CRY2PHR/CIBN light-inducible system with split protease. In the dark, the CRY2PHR and CIBN proteins do not interact (top), while illumination with blue light results in heterodimerization and reconstitution of split protease (bottom), which in turn cleaves the target protein. Activity of PPV protease were analyzed by Western blot. HEK293T cells were transfected with 1:3 ratio of plasmids coding for CRY2PHR:CIBN with split PPV protease. 24 hours after transfection, cells were illuminated with blue light at 460nm for indicated periods of time. Cells were immediately lysed and the cleaved products were analysed by Western blot using anti-AU1 antibodies.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 556
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 142
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 592
Illegal BsaI.rc site found at 772
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