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Revision as of 18:11, 18 October 2016
Protease orthogonality
The first challenge in construction of a new protease-based signaling cascade was the selection of appropriate proteases. The candidate proteases should: i) recognize defined target cleavage sequences, preferably as long as possible; ii) be active in mammalian cells, but not toxic to them and iii) be inducible, ideally through the reconstitution of split protein fragments. Most importantly, a large number of proteases with similar properties but different substrates should be available to allow for modular construction of signaling pathways and logic functions. These proteases should be orthogonal to each other, meaning that their specific cleavage sites should not be recognized by other proteases in the system.
We found that the tobacco etch virus protease (TEVp) was the only protease described in the literature to match our criteria.
TEV protease is a highly specific, 242 amino acids long, 27 kDa cysteine
protease, that originates from the tobacco etch virus (TEV) of the Potyvirus
genus.
It has a target recognition sequence of seven amino acids, ENLYFQ-S/G, where
cleavage occurs after the glutamine residue (denoted by the – symbol),
and the final residue of the recognition sequence can be either S or G (denoted
by the / symbol). This substrate sequence is scarcely represented in the
proteome. TEV protease is therefore relatively non-toxic
Despite its widespread use in biotechnology, TEVp also displays some
shortcomings, the most prominent of them being self-cleavage. Substitution
of Ser-219 with Val or Pro
To overcome the lack of inducible orthogonal proteases, we looked for the characterized TEVp mutants and naturally occurring proteases, closely related to TEVp that might also be used to function as split proteases.
Results
TEVp variants
Based on the sequence alterations described by Yi et al.
Yi et al.
To test these two proteases we used a cleavable firefly luciferase (fLuc) reporter with an appropriate cleavage sequence inserted in a permissible site. We observed a significant decrease in luciferase activity upon co-expression of the reporters and their corresponding proteases, whereas co-expression of an orthogonal protease did not decrease the luciferase activity ( 1 ). These results were additionally confirmed by results from western blotting, where cleavage of the luciferase reporter was detected only in the presence of the corresponding protease, but not in the presence of orthogonal proteases ( 2 ).
HEK293T cells were transfected with the indicated fLuc:TEVs and TEVp variant constructs. Luciferase activity was determined 24 h after transfection.
No data has previously been reported on TEVpE and TEVpH toxicity, therefore we expressed each TEVp variant in HEK293T cells and performed a viability assay. Even after transfection with a high amount of the plasmid for each respective protease, the cells showed high viability, with practically no difference when compared to cells transfected with a mock plasmid ( 3 ).
HEK293T cells were transfected with 2000 ng of the indicated protease and 500 ng of the indicated reporter. Cells were lysed and analyzed by western blotting with anti-AU1 antibodies. The cleaved reporter (55 kDa) was detected only in the presence of the corresponding TEVp variant.
HEK293T cells were transfected with plasmids coding for different TEVp variants. 48 hours after transfection, cells were stained with the trypan blue dye, which stains only dead cells. The ratio of live and dead cells was analysed with the Countess automated cell counter.
TEVp homologs
Introduction of two new TEVp variants expanded our repertoire of tools, demonstrating that we can use the results of mutational screenings for protease variants. However, a larger number of strictly orthogonal proteases would be required for modular design of complex logic circuits. We therefore decided to investigate the activity of de novo created split proteases from the potyviridae family.
The NIa proteases from the potyviridae group of plant viruses in general
recognize a seven amino acid sequence motif as their substrate. They are
are classified as cysteine proteases with an active site closely related to
eukaryotic serine proteases. The NIa proteases adopt a characteristic
two-domain antiparallel β-barrel fold. The active site of the protease
comprises a catalytic triad: His-46, Asp-81, Cys-151 (amino acids numbered
according to the TEVp sequence) with a Gly-x-Cys-Gly motif around the active
cysteine residue
We searched for different potyviruses available on UniProt, paying particular attention to any evidence of orthogonality among their target substrates. We decided to test the plum pox virus protease (PPVp), the soybean mosaic virus protease (SbMVp), and the sunflower mild mosaic virus protease (SuMMVp).
Homology models obtained from SWISS-MODEL using the crystal structure of TEVp (red) (PDB code: 1LVB) as template. The homology models of the potyviridae family proteases PPVp (blue), SbMVp (cyan) and SuMMVp (yellow) are shown in ribbons.
PPVp is one of the most studied potyviral proteases after the TEV protease.
Its substrate (PPVs) has an amino acid sequence NVVVHQ-A.
In contrast to TEVp, it has been reported that PPVp is resistant to
self-cleavage at the C-terminus
SbMVp has been
recently studied by Seo et al. as a tool for protein-protein interaction
studies in the soybean plant. The substrate (SBMVs) has been determined to
be the
sequence ESVSLQ-S
Similarly, SuMMVp has been used by Fernandez-Rodriguez et al.
All selected potyviral proteases were designed as synthetic genes and their activity was tested in mammalian cells using cyclic luciferase reporters, which propagate luciferase activity only upon cleavage. We detected an increase of luciferase activity only in the corresponding protease-reporter pairs, confirming exquisite orthogonality of the selected proteases and their activity in the human cell chassis ( 5 ).
HEK293T cells were transfected with the indicated cycLuc reporters and proteases. Luciferase activity was detected only in the presence of a protease and cycLuc reporter containing appropriate protease cleavage site.
References
