Part:BBa_K1965042

cycLuc_PPVs

Introduction

The cyclic luciferase system enhances the circularly permuted luciferase (cpLuc) ^{[3, 4]} by fusing two fragments of an intein to the ends of cpLuc ^{[1, 2]}. Inteins are protein fragments that allow protein splicing and cyclization by formation of a new peptide bond between the N- and C-termini of the protein. We expected this reporter to result in a higher signal due to the stabilization of the protein by cyclization (1). To further optimize the dynamic range of the system, a PEST sequence for fast degradation of the protein was included at the C-terminus of the protein. This sequence targets any of the unspliced protein to degradation, while the spliced cyclic protein remains stable, since the PEST sequence is excised along with the intein fragments during the splicing reaction ^{[1, 2]}.

Sheme of cycLuc structure, splicing and activation by protease cleavage.
text

Cyclic luciferase with the PPVs sequence (cycLuc_PPVs) contains the circularly permutated firefly luciferase, flanked by intein sequences. The two parts of fLuc are connected through the PPVs recognition sequence (NVVVHQ-A).

Characterization

This construct was expressed under the CMV promoter and used for testing the orthogonality of different TEVp homologs and studying the activity split PPVp by measuring the activity of fLuc. The coding sequence for cycLuc_PPVs was deposited in pSB1C3.

For testing the orthogonality, HEK293T cells were cotransfected by the plasmids with the whole protease and cycLuc reporters as shown on 2

Protease orthogonality
HEK293T cells were transfected with 100 ng of cycLuc_PPVs reporter and 70 ng of the indicated proteases. Luciferase activity was detected only in the presence of the cycLuc_PPVs reporter and PPVp.

To test the activity of split proteases, HEK293T cells were cotransfected by the plasmids with rapamycin inducible split protease and corresponding cycLuc reporter as shown on 3.

Activitiy of split PPVp based on rapamycin inducible system.
HEK293T cells were trasnfected with 100 ng of the cycLuc_PPVs reporter and 70 ng of each split PPVp fragment. The whole PPVp (70 ng) was used as positive control. An increase in luciferase activity was detected in cells induced with rapamycin.

References

^[1]Kanno A., Yamanaka Y., Hirano H., Umezawa Y., Ozawa T. 2007. Cyclic Luciferase for Real-Time Sensing of Caspase-3 Activities in Living Mammals.
^[2]Kanno A., Umezawa Y., Ozawa, T. 2009. Detection of Apoptosis Using Cyclic Luciferase in Living Mammals.
^[3]Fan, F., Binkowski, B. F., Butler, B. L., Stecha, P. F., Lewis, M. K., & Wood, K. V. (2008). Novel genetically encoded biosensors using firefly luciferase. ACS Chemical Biology, 3(6), 346–351. https://doi.org/10.1021/cb8000414
^[4]Wigdal, S. S., Anderson, J. L., Vidugiris, G. J., Shultz, J., Wood, K. V, & Fan, F. (2008). A novel bioluminescent protease assay using engineered firefly luciferase. Current Chemical Genomics, 2, 16–28. https://doi.org/10.2174/1875397300802010016

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1090
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 865
  Illegal NgoMIV site found at 886
  Illegal NgoMIV site found at 1114
  Illegal NgoMIV site found at 1177
  Illegal NgoMIV site found at 2239
  Illegal AgeI site found at 589
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 771