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.
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.
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 (
3A and B). The insertion site for the substrate sequence was based on the
previously described split luciferase system
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,
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
The circularly permutated luciferase makes use of this requirement for a
conformational change by rearranging the sequence of the protein. The permutation is
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
(3C and D), which contains a protease cleavage site
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.
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
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.
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