Split luciferase

Ozawa, T., Kaihara, A., Sato, M., Tachihara, K., and Umezawa, Y. (2001) Split luciferase as an optical probe for detecting protein-protein interactions in mammalian cells based on protein splicing. Anal. Chem. 73, 2516-2521. 

Fig. 1.12 Mechanism of the bioluminescence reaction of firefly luciferin catalyzed by firefly luciferase. Luciferin is probably in the dianion form when bound to luciferase. Luciferase-bound luciferin is converted into an adenylate in the presence of ATP and Mg2+, splitting off pyrophosphate (PP). The adenylate is oxygenated in the presence of oxygen (air) forming a peroxide intermediate A, which forms a dioxetanone intermediate B by splitting off AMP. The decomposition of intermediate B produces the excited state of oxyluciferin monoanion (Cl) or dianion (C2). When the energy levels of the excited states fall to the ground states, Cl and C2 emit red light (Amax 615 nm) and yellow-green light (Amax 560 nm), respectively.

Fig. 6.3.5 A reaction scheme proposed by Tsuji (2002) for the Watasenia bioluminescence. The proposed mechanism involves the adenylation of luciferase-bound luciferin by ATP, like in the bioluminescence of fireflies. However, the AMP group is split off from luciferin before the oxygenation of luciferin, differing from the mechanism of the firefly bioluminescence. Thus the role of ATP in the Watasenia bioluminescence reaction remains unclear. Reproduced with permission from Elsevier.

Fig. 8. Protein-protein interaction study based on split intein. In order to monitor the protein interaction in vivo, the N- and C-terminal halves of the intein (N-intein and C-intein) are fused to N- and C-terminal halves of EGFP (A), or luciferase (B). Each of these fusion proteins is linked to the protein of interest (protein A) and its target protein (protein B). Upon protein A-protein B cooperation, the closely oriented intein fragments mediate intein splicing. The measurement of fluorescence intensity originated from the reconstituted mature EGFP protein or measurement of luciferase luminescence is possible.

Figure 1(a). A schonatic diagram of the split Renilla luciferase complementation strategy (b). Sdiematk rqtresoitation of the plasmid construas... 

Figure 2. Luminescence image of ttie cells expressing/feni/Ja luciferase (hRL124C/A) Efficiency of split Renilla luciferase complementation...

Figure 3(a). Amino-acid sequence of Renilla luciferase and split positions (b). Luminescence analysis of split Renilla lucifo ase Aision proteins forefficieiit complementation... 

Kaihara A, Kawai Y, Sato M, Ozawa T, Umezawa Y. Locating a protein-protein interaction in living cells via split Renilla luciferase complementation. Anal Biochem 2003 75 4176-81. 

Asami Kaihara (Department of Chemistry, School of Science, University of Tokyo, Tokyo, Japan) Flashing a protein-protein interaction in living cells by a split Renilla luciferase complementation... 

Figure 12 In vitro evaluation of the lipidoid library for sIRNA delivery, (a) Percent reduction of firefly luciferase expression in HeLa cells with lipidoid/ siRNA formulations. The data are split into five groupings spanning 0-100% luciferase reduction for ease of analysis, (b) Optimal knockdown levels in HeLa determined by screening at four different lipidoid/siRNA ratios, (c-e) Dose response silencing in HeLa (c), HepG2 (d), and primary macrophage (e) cells. Reprinted from Akinc, A. Zumbuehl, A. Goldberg, M. et at. Nat. Biotechnol. 2008, 26 (5), 561-569, with permission from the Nature Publishing Group.

Paulmurugan, R., Gambhir, S. (2003). Monitoring protein-protein interactions using split synthetic reniHa luciferase protein-fragment-assisted complementation. Analytical Chemistry, 75, 1584—1589. 

Paulmurugan R, Gambhir S. Monitoring protein -protein interactions using split synthetic Renilla luciferase protein-fragment-assisted complementation. Anal Chem 2003 75 1584-1589. 

It is important to note, however, that flavoquinone is protonated at N(l) (and not at N(5)) with a pK as low as zero (21). This results in the coincidence of the 450 and 370 nm transitions of Flox to form a single band centered at 395 nm which has an extinction of >20.000 cm" It is a main characteristic of covalently (protein) bound flavocoenzymes (see chapter below), that this band remains split ( max 375 nm, shoulder 5 410) as a consequence of 8a-substi-tution. The cations 1-RFlox are fluorescent with an emission around 490 nm, provided R is kinetically stable over the lifetime of the excited state. This is trivially true for R = alkyl, but it can be reached also for R = H in rigid media (28). In flavin-dependent bioluminescence of photobacteria, l-RFl x (and certainly not Fl x) is most probably the emitting species, while it remains to be proved whether R in the luciferase ... 

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