Reporters

Three luciferase based reporters for detection of protease activity were designed and tested.
Cleavable firefly luciferase was used as an inverse reporter, displaying a decrease in luminescence upon proteolytic cleavage.
Circularly permuted and cyclic luciferase were used as direct reporters, displaying an increase in luminescence upon proteolytic cleavage.
Fluorescent and enzymatic reporters with cleavable ER retention signals were designed to test induced protein secretion.

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

To measure the activity of the proteases, we used three types of reporters based on firefly luciferase: the cleavable fLuc inverse reporter, the circularly permutated fLuc (cpLuc) and the cyclic fLuc (cycLuc).

Luciferase reporters for detection of proteolytic activity can be designed to lead either to a decrease of its activity due to proteolysis (inverse reporter) or to increase luciferase activity by proteolytic cleavage of an inactive enzyme. The cleavable luciferase inverse reporter assay is expected to be relatively insensitive as a large fraction of the luciferase, typically more than 20%, has to be degraded before an appreciable difference in the output is detected, while an assay that leads to activation of luciferase might be able to detect much proteolytic activity.

**Luciferase reporters.**

(A) Scheme of wild-type luciferase. The site of insertion for the cleavage peptide is marked by an arrow. (B) Scheme of cleavable luciferase and the mechanism of inactivation by proteolysis. (C) Scheme of wild-type luciferase. The split site and the circular permutation are marked by arrows. (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.

Additionally, we developed a split luciferase system that functions as an output for logic functions, integrating the activity of orthogonal proteases. Finally, to measure protein retention and release from the endoplasmic reticulum (ER), we used TagRFP and SEAP reporters.

nbsp;Results

nbsp;Cleavable luciferase

We inserted amino acid sequences targeted by selected proteases into a loop of the firefly luciferase. The substrate sequence thus divided the fLuc into two fragments (nLuc and cLuc), with a protease cleavage site between them ( 3 A 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, in turn decreasing the fLuc activity. In this system higher protease activity corresponds to a lower luciferase activity. The reporters were additionally equipped with protein tags at the N- and C-termini in order to allow immunostaining for detection of protein cleavage by western blotting (Light-dependent mediator Figure 3).

nbsp;Circularly permuted luciferase

The first reporter to measure protease activity that results in generation of luciferase activity by 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 100 ng of indicated reporter constructs and 70 ng of their corresponding proteases. Luciferase activity was measured 24 h 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 ( 3 C and D), which contains a protease cleavage site Fan2008 . This linker prevents the conformational change required for efficient catalysis; therefore, the catalytic cycle is triggered only upon cleavage of the linker by a protease.

We designed and tested four new cpLuc reporters. Activity of all four reporters depended on cleavage by the corresponding protease as expected ( 2 ), however light emission from this reporter system was relatively low, compelling us to look for a more efficient reporter.

nbsp;Cyclic luciferase

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 ( 3 E). 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 .

nbsp;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 (Protease inducible secretion Figure 1.

nbsp;SEAP

For a protein secretion assay with increased sensitivity we replaced the TagRFP with the 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 the Quanti-blue colorimetric enzyme assay (Invivogen) (Protease inducible secretion Figure 2).

Team:Slovenia/Protease signaling/Reporters

nbsp;Protease reporters