Part:BBa_K1965045

cLuc:A:HA

This construct consists of the C-terminal part of the split luciferase attached to the coiled coil A. The sequence for the coiled coil A was taken from the Shekhawat et al. (1). The construct also features a HA protein tag on the C-terminus. Coiled coil A dimerizes with the coiled coil B, which was also described in Shekhawat et al.. If the B coil is fused to the N-terminal part of the split luciferase (BBa_K1965046), the luciferase can reassemble and regain its activity through the dimerization of the coiled coil pair A and B (2A) ^[1] .

Sequence alignment of different coiled coils used to tune the affinity of antiparallel coiled-coils.
We designed different destabilized coils from our coiled coil pair AP4 and P3; Ile and Leu were substituted with Ala at the a and/or d position of the first and/or second heptad of P3mS (a more soluble variant of the original P3).

Interactions and protease activated AB coiled-coil formation.
HEK293T cells were transfected with plasmids, coding for the appropriate coiled coil constructs. 24 h after transfection cells were lysed and luciferase activity was determined with the dual luciferase assay. (A) A and B coiled-coils, fused the split firefly luciferase spontaneously interact and reconstitute firefly luciferase. (B) A’:TEVs:B:nLuc and cLuc:A:PPVs:B’2A auto-inhibitory coiled coils reconstitute activity of firefly luciferase upon cleavage by TEV and PPV proteases. Successive luciferase reconstitution is observed only at high amounts of plasmids coding for the A’:TEVs:B:nLuc and cLuc:A:PPVs:B’2A constructs.

^[1] Shekhawat, S. S., Porter, J. R., Sriprasad, A., & Ghosh, I. (2009). An Autoinhibited Coiled-Coil Design Strategy for Split-Protein Protease Sensors. Jacs, 131(42), 15284–15290.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]