Difference between revisions of "Team:Slovenia/Protease signaling/Orthogonality"
| Line 87: | Line 87: | ||
<p>We found that the tobacco etch virus protease (TEVp) was the only protease described in the literature to match our criteria.</p> | <p>We found that the tobacco etch virus protease (TEVp) was the only protease described in the literature to match our criteria.</p> | ||
| + | <div class="ui styled fluid accordion"> | ||
| + | <div class="title"> | ||
| + | <i class="dropdown icon"></i> | ||
| + | Further explanation ... | ||
| + | </div> | ||
| + | <div class="content"> | ||
| + | <p>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 and is 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<x-ref>Parks1994</x-ref> and can be safely expressed in host cells. Due to this non-toxicity and | ||
| + | its high cleavage specificity, TEVp is an attractive protease for use in several biotechnological applications, such as the removal of the affinity tags | ||
| + | from recombinant proteins. | ||
| + | </p> | ||
| + | <p>Despite its widespread use in the biotechnology, TEVp also displays some shortcomings, the most prominent of them being self-cleavage. Substitution | ||
| + | of Ser-219 with Val or Pro <x-ref>Cesaratto2015</x-ref> or a replacement of the C-terminal sequence MSELVYSQ with the sequence MNEGGGLE | ||
| + | <x-ref>Cesaratto2015</x-ref> decreased the self-cleavage of TEVp and thereby increased its activity. | ||
| + | </p> | ||
| + | </div> | ||
| + | </div><br/> | ||
| + | <p>To overcome this 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.</p> | ||
| + | |||
| + | <h3>TEVp variants</h3> | ||
| + | <p>Based on the sequence alterations described by Yi et al. <x-ref>Yi2013</x-ref> our team decided to test the two variants of TEVp - TEVpE and TEVpH.</p> | ||
| + | |||
| + | |||
| + | |||
</div> | </div> | ||
Revision as of 11:57, 16 October 2016
nbsp;Orthogonality of proteases
The first challenge in the construction of a new protease-based signaling cascade was the selection of appropriate proteases. The candidate proteases should recognize defined target cleavage sequences, preferably as long as possible; they should be active in mammalian cells, but not toxic to them and inducible, ideally through the reconstitution of split protein fragments. Most importantly, a large number of proteases with similar properties but with different specificities should be available to allow for modular construction of signaling pathways and logic functions and these proteases should be orthogonal to each other, meaning they should have specific cleavage sites not recognized by the 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 and is 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 the biotechnology, TEVp also displays some shortcomings, the most prominent of them being self-cleavage. Substitution
of Ser-219 with Val or Pro
To overcome this 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.
TEVp variants
Based on the sequence alterations described by Yi et al.
