Team:Slovenia/Protease signaling/Reporters

Reporters

As our project was aimed develop novel orthogonal signaling pathways based on proteases, as well as at the development of the protein ER retention and release system, we tested and adapted several types of reporters, that will be useful for other iGEM teams.

To measure the activity of the proteases we used three types of reporters based on the firefly luciferase: the cleavable fLuc, the circularly permutated fLuc (cpLuc) and the cyclic fLuc (cycLuc). Additionally, we developed a split luciferase system that functions as an output of logic gates, which integrated the activity of orthogonal proteases.

Finally, to measure the ER protein retention and release, we used TagRFP and SEAP reporters.

Luciferase reporter of the proteolytic activity can be designed either to lead to the decrease of its activity due to proteolysis or to generate the activity by cleavage. Cleavable luciferase assay is expected to be relatively insensitive as it can only detect if a large fraction of the luciferase has been degraded, typically more than 20%, while an assay that leads to the activation of the luciferase might be able to detect much smaller fraction of the proteolytic cleavage.

Cleavable luciferase

Into the loop of the firefly luciferase (fLuc) we inserted amino acid sequence that is targeted by proteases. The substrate sequence thus divided the fLuc into two fragments (nLuc and cLuc), with a protease cleavage site between them (1A and B). The insertion site for the substrate sequence was based on the previously described split luciferase system Shekhawat2009, where we expected that this site would also be permissible to short linker insertion without significantly altering luciferase activity. Upon addition of an appropriate protease, the reporter would be cleaved at the substrate site and the two fragments would dissociate and in turn decrease the fLuc activity. In this system higher protease activity corresponds to a lower luciferase activity. The reporters were additionally equipped with a protein tag at the N-terminal and C-terminal end in order to allow immunostaining to detect protein cleavage by the western blot (1).

Circularly permuted luciferase

The first reporter to measure protease activity that results in the generation of the luciferase activity by the proteolytic cleavage was a circularly permuted version of the firefly luciferase (cpLuc). Luciferase is an oxidative enzyme that produces bioluminescence. The protein consists of two compact domains: the larger N- and the smaller C-terminal domain. The C-terminal domain is connected to the N-terminal domain by a flexible hinge. When bound to the substrate luciferin, luciferase has to undergo a conformational change from an open to a closed form with the two domains coming together to enclose the substrate and efficiently catalyze its oxidation Conti.

**Proteolytic activity determined by the cpLuc reporters.**
HEK293T cells were transfected with 100ng of indicated reporter constructs and 70ng of their corresponding proteases. Luciferase activity was measured 24h after transfection. The results are presented as normalized firefly luciferase activity (RLU).

The circularly permutated luciferase makes use of this requirement for a conformational change by rearranging the sequence of the protein. The permutation is obtained by placing the C-terminal region of the protein (amino acids 234-544) upstream of the N-terminal region (amino acids 4-233) and connecting them by a short linker (1C and D), which contains a protease cleavage site Fan2008. This linker prevents the conformational change required for the efficient catalysis; therefore, the catalytic cycle is triggered only upon cleavage of the linker by a protease.

Activity of the cpLuc depended on the protease cleavage for all four tested proteases as expected (2), however light emission from this reporter system was relatively low, compelling us to look for another reporter.

Cyclic luciferase

**Luciferase reporters.**
(A) Scheme of wild-type luciferase showing the site of insertion for the cleavage peptide (B) Scheme of cleavable luciferase and the mechanism of inactivation by proteolysis (C) Scheme of wild-type luciferase showing the split site and the circular permutation (D) Scheme of circularly permuted luciferase and the mechanism of activation by proteolysis. (E) Scheme of cyclic luciferase, cyclization by intein excision and mechanism of activation by proteolysis.

The cyclic luciferase system takes the mechanism of cpLuc one step further by fusing two fragments of an intein to the ends of the cpLuc. 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, which generates an inactive protein that can be activated by the proteolytic cleavage. We expected this reporter to result in a higher signal from the cpLuc due to the stabilization of the protein by cyclization (3E). To further optimize the dynamic range of the system, a PEST sequence for fast digestion 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 Kanno2007.

TagRFP

For detection of the localization of our ER targeted reporters inside cells we used the fluorescent protein TagRFP (Evrogen), which was fused to the appropriate localization and proteolytic target tags and used confocal microscopy for detection. Merzlyak et al. Merzlyak2007, modified the wild type RFP from the sea anemone Entacmaea quadricolor to prolong its fluorescence lifetime and make it less susceptible to pH. With addition of the AU1 tag, we were also able to detect its expression and secretion with western blot (4).

SEAP

For an assay of protein secretion with increased sensitivity we replaced the TagRFP with human secreted alkaline phosphatase (SEAP) reporter, which can readily and accurately be quantified within the medium of transfected cells Berger1988. SEAP released into the medium was detected with Quanti-blue colorimetric enzyme assay (Invivogen) (5).