Difference between revisions of "Team:Slovenia/Protease signaling/Orthogonality"

 
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                                     site. We observed a significant decrease in luciferase activity upon
 
                                     site. We observed a significant decrease in luciferase activity upon
 
                                     co-expression of the reporters and their corresponding proteases, whereas
 
                                     co-expression of the reporters and their corresponding proteases, whereas
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                                     co-expression of an orthogonal protease did not decrease the luciferase activity (
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                                     co-expression of an orthogonal protease did not decrease the luciferase activity (<ref>1</ref>). These results were additionally confirmed by results from western blotting, where
−
                                    <ref>1</ref>
+
−
                                    ). These results were additionally confirmed by results from western blotting, where
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                                     cleavage of the luciferase reporter was
 
                                     cleavage of the luciferase reporter was
 
                                     detected only in the presence of the corresponding protease, but not in the presence
 
                                     detected only in the presence of the corresponding protease, but not in the presence
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                                     of orthogonal proteases (
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                                     of orthogonal proteases (<ref>2</ref>).
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                                    <ref>2</ref>
+
−
                                    ).
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                                 </p>
 
                                 </p>
 
                                 <div style="float:left; width:100%">
 
                                 <div style="float:left; width:100%">
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                                         <img src="https://static.igem.org/mediawiki/2016/8/87/T--Slovenia--4.4.1.png">
 
                                         <img src="https://static.igem.org/mediawiki/2016/8/87/T--Slovenia--4.4.1.png">
 
                                         <figcaption><b>Activity and orthogonality of TEVp variants.</b><br/>
 
                                         <figcaption><b>Activity and orthogonality of TEVp variants.</b><br/>
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                                             <p style="text-align:justify">HEK293T cells were transfected with the
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                                             <p style="text-align:justify">(A) Activity of cleavable fLuc with the wild type TEVs cleavage sequence is reduced only in the presence of wild type TEVp. (B) Activity of cleavable fLuc with the TEVsE cleavage sequence is reduced only in the presence of TEVpE. (C) Activity of cleavable fLuc with the  TEVsH cleavage sequence is reduced only in the presence of TEVpH. HEK293T cells were transfected with the
 
                                                 indicated fLuc:TEVs and TEVp variant constructs. Luciferase activity was
 
                                                 indicated fLuc:TEVs and TEVp variant constructs. Luciferase activity was
−
                                                 determined 24 h after transfection.
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                                                 determined 24 h after transfection. (D) Table of cleavage sequences for the wild type TEVp and its variants. The cleavage occurs before the final Ser and is indicated by -.
 
                                             </p>
 
                                             </p>
 
                                         </figcaption>
 
                                         </figcaption>
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                                     amount of the plasmid for each respective protease, the cells showed high viability,
 
                                     amount of the plasmid for each respective protease, the cells showed high viability,
 
                                     with practically
 
                                     with practically
−
                                     no difference when compared to cells transfected with a mock plasmid (
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                                     no difference when compared to cells transfected with a mock plasmid (<ref>3</ref>).
−
                                    <ref>3</ref>
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−
                                    ).
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                                 </p>
 
                                 </p>
  
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                                 <br/>
 
                                 <br/>
  
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                                 <p>We searched for different potyviruses available on UniProt, paying particular
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                                 <p>We searched for different potyviruses available on UniProt (<ref>4</ref>), paying particular
 
                                     attention to any evidence of orthogonality among their target substrates.
 
                                     attention to any evidence of orthogonality among their target substrates.
 
                                     We decided to test the plum pox virus protease (PPVp), the soybean mosaic virus
 
                                     We decided to test the plum pox virus protease (PPVp), the soybean mosaic virus
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                                             <p style="text-align:justify">Homology models obtained from <a style="padding-right: 0"
 
                                             <p style="text-align:justify">Homology models obtained from <a style="padding-right: 0"
 
                                                     href="https://swissmodel.expasy.org/">SWISS-MODEL</a> using the
 
                                                     href="https://swissmodel.expasy.org/">SWISS-MODEL</a> using the
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                                                 crystal structure of TEVp (red) (PDB code: 1LVB) as template. The
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                                                 crystal structure of TEVp (red) (<a style="padding-right: 0"
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                                                    href="http://www.rcsb.org/pdb/explore.do?structureId=1LVB">PDB 1LVB</a>) as template. The
 
                                                 homology models of the potyviridae family proteases PPVp (blue), SbMVp
 
                                                 homology models of the potyviridae family proteases PPVp (blue), SbMVp
 
                                                 (cyan) and
 
                                                 (cyan) and
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                                     protease-reporter pairs, confirming exquisite orthogonality of the selected
 
                                     protease-reporter pairs, confirming exquisite orthogonality of the selected
 
                                     proteases and
 
                                     proteases and
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                                     their activity in the human cell chassis (
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                                     their activity in the human cell chassis (<ref>5</ref>).
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                                    <ref>5</ref>
+
−
                                    ).
+
 
                                 </p>
 
                                 </p>
 
                                 <div style="clear:right; width:70%">
 
                                 <div style="clear:right; width:70%">
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                                         <img src="https://static.igem.org/mediawiki/2016/8/83/T--Slovenia--4.4.4.png">
 
                                         <img src="https://static.igem.org/mediawiki/2016/8/83/T--Slovenia--4.4.4.png">
 
                                         <figcaption><b>Protease orthogonality.</b><br/>
 
                                         <figcaption><b>Protease orthogonality.</b><br/>
−
                                             <p style="text-align:justify">HEK293T cells were transfected with the
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                                             <p style="text-align:justify">(A) Table of cleavage sequences for TEVp and its homologues. The cleavage occurs before the final Ser or Ala and is indicated by -. (B) Heat map showing orthogonality of the TEVp homologues. Dark red corresponds to higher luciferase activity. (C) Orthogonality of TEVp homologues shown as bar graphs. HEK293T cells were transfected with the
 
                                                 indicated cycLuc reporters and proteases. Luciferase activity was
 
                                                 indicated cycLuc reporters and proteases. Luciferase activity was
 
                                                 detected only in the presence
 
                                                 detected only in the presence
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                             </div>
 
                             </div>
 
</div>
 
</div>
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                         <h1 class="ui left dividing header"><span id="ref-title" class="section colorize">&nbsp;</span>References
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                         <h3 class="ui left dividing header"><span id="ref-title" class="section colorize">&nbsp;</span>References
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                         </h1>
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                         </h3>
 
                         <div class="ui segment citing" id="references"></div>
 
                         <div class="ui segment citing" id="references"></div>
 
</div>
 
</div>

Latest revision as of 13:56, 19 October 2016

Orthogonality

Reporters Orthogonality Achievements Introduction TEVp variants TEVp homologs Split proteases

 Protease orthogonality

  • Two TEVp variants (TEVpE and TEVpH) were tested and proved to be mutually orthogonal.
  • Upon overexpression none of the tested proteases were toxic to mammalian cells, demonstrating that they do not interfere with essential cellular processes.
  • Additionally, three TEVp homologues (PPVp, SbMVp and SuMMVp) were tested and proved to be fully orthogonal.

 

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.

Further explanation ...

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 Parks1994 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 removal of affinity tags from recombinant proteins.

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 Cesaratto2015 or a replacement of the C-terminal sequence MSELVYSQ with the sequence MNEGGGLE Cesaratto2015 decreased the self-cleavage of TEVp and thereby increased its activity.


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. Yi2013 , our team decided to test two mutated variants of TEVp - TEVpE and TEVpH.

Further explanation ...

Yi et al. Yi2013 tackled the problem of acquiring novel orthogonal proteases by screening a library of TEVp mutants for orthogonal specificity. They designed a novel yeast ER sequestration screening assay that allowed them to identify variants of TEVp that recognize alternative substrates. The two most prominent mutants were TEVpE, which cleaves the substrate ENLYFE-S (TEVsE) and TEVpH, cleaving the substrate ENLYFH-S (TEVsH). Although the two variants were also able to cleave the wild type TEVp substrate ENLYFQ-S (TEVs), they displayed a high preference for their own variation of the substrate and were mutually orthogonal.


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).

Activity and orthogonality of TEVp variants.

(A) Activity of cleavable fLuc with the wild type TEVs cleavage sequence is reduced only in the presence of wild type TEVp. (B) Activity of cleavable fLuc with the TEVsE cleavage sequence is reduced only in the presence of TEVpE. (C) Activity of cleavable fLuc with the TEVsH cleavage sequence is reduced only in the presence of TEVpH. HEK293T cells were transfected with the indicated fLuc:TEVs and TEVp variant constructs. Luciferase activity was determined 24 h after transfection. (D) Table of cleavage sequences for the wild type TEVp and its variants. The cleavage occurs before the final Ser and is indicated by -.

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).

Orthogonality and activity of TEVpE and TEVpH.

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.

Different TEVp variants are not toxic to HEK293T cells.

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.

Further explanation ...

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 Yoon2000, Nunn2005 .


We searched for different potyviruses available on UniProt (4), 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).

Orthogonal proteases from the potyviridae family.

Homology models obtained from SWISS-MODEL using the crystal structure of TEVp (red) (PDB 1LVB) as template. The homology models of the potyviridae family proteases PPVp (blue), SbMVp (cyan) and SuMMVp (yellow) are shown in ribbons.

Further explanation ...

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 Zheng2008, Garcia1991 .
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 Seo2016, Yoon2000 .
Similarly, SuMMVp has been used by Fernandez-Rodriguez et al. Fernandez-Rodriguez2016 . in a study of genetic circuits using potyviral proteases. The substrate (SuMMVs) has been defined as the sequence EEIHLQ-S/G Fernandez-Rodriguez2016 .


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).

Protease orthogonality.

(A) Table of cleavage sequences for TEVp and its homologues. The cleavage occurs before the final Ser or Ala and is indicated by -. (B) Heat map showing orthogonality of the TEVp homologues. Dark red corresponds to higher luciferase activity. (C) Orthogonality of TEVp homologues shown as bar graphs. 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