Coding sequence of FKBP-Cluc394
FKBP-C-luc394 fusion protein ORF
Firefly (Photinus pyralis) luciferase can be split to N-terminal (N-luc) and C-terminal (C-luc) fragments and each of them is inactive. When they two reassembled non-covalently, the enzymatic activity would be reconstituted and the recovered luciferase is able to oxidize luciferin and produce detectable bioluminescence. Currently there are different combinations of split fragments, among which N-luc416/ C-luc398 and N-luc398/ C-luc394 are widely used.
FKBP is a monomeric and highly abundant cytosolic protein that serves as the primary receptor for the immunosuppressive ligands FK506 and rapamycin. Previously Raik Gruenberg had already designed the part BBa_J18925, containing the coding sequence of FKBP. Rapamycin-binding domain (FRB) of human mTOR (mammalian Target of Rapamycin) binds with high affinity to FKBP. Rapamycin is able to induce the dimerization to form a FRB-rapamycin-FKBP complex. This protein-protein interaction can be visualized by split luciferase. FRB and FKBP are fused to N-luc and C-luc respectively, and adding rapamycin can induce the approaching and reconstitution of split luciferase (Figure 1a).
2015 Peking iGEM improved the previous part BBa_J18925, they fused C-luc394 to C terminus of FKBP (FKBP-C-luc394, BBa_K1689006) and combined it with N-luc398-FRB (BBa_K1689004) to validate the functional reconstitution of split luciferase. However, compared with N-luc416/ C-luc398, the bioluminescence intensity didn't increase significantly after rapamycin was added (Figure 1). Therefore we discarded them and chose N-luc416/ C-luc398 as our split luciferase in the project (See BBa_K1689003 or BBa_K1689005.)
Figure 1. Rapamycin-induced N-luc-FRB/ FKBP-C-luc complementation. (a) The working mechanism of rapamycin induced dimerization. The interacting protein partners (FRB & FKBP) get closer and dimerize soon after rapamycin is added (40nM) , thus to reconstitute the enzymatic activity of luciferase. (b) The experimental data. Error bars denote s.d.; n=3.
1. Ramasamy Paulmurugan, Sanjiv S. Gambhir. Firefly Luciferase Enzyme Fragment Complementation for Imaging in Cells and Living Animals. Anal Chem. 2005 March 1; 77(5): 1295–1302.
2. Rivera, V. M., T. Clackson, S. Natesan et al. A humanized system for pharmacologic control of gene expression. Nat. Med. 1996. 2:1028–1032.
3. Ramasamy Paulmurugan, Sanjiv S. Gambhir. Combinatorial Library Screening for Developing an Improved Split-Firefly Luciferase Fragment-Assisted Complementation System for Studying Protein-Protein Interactions. Anal. Chem. 2007, 79, 2346-2353.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12Illegal NheI site found at 7
Illegal NheI site found at 30
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25Illegal NgoMIV site found at 662
Illegal AgeI site found at 801
- 1000COMPATIBLE WITH RFC