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

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<div class="main ui citing justified container">
 
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<div>
 
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<h1 class = "ui left dividing header"><span id = "ach" class="section">&nbsp;</span>Protease orthogonality - in progress TL</h1>
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<h1 class = "ui left dividing header"><span id = "ach" class="section">&nbsp;</span>Protease orthogonality</h1>
 
<div class = "ui segment" style = "background-color: #ebc7c7; ">
 
<div class = "ui segment" style = "background-color: #ebc7c7; ">
 
<p><b><ul>
 
<p><b><ul>
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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
 
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  
 
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  
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its high cleavage specificity, TEVp is an attractive protease for use in several biotechnological applications, such as the removal of the affinity tags
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its high cleavage specificity, TEVp is an attractive protease for use in several biotechnological applications, such as removal of affinity tags
 
from recombinant proteins.  
 
from recombinant proteins.  
 
</p>
 
</p>
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</div>
 
</div>
 
 
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<p style="clear:left;">No data has previously been reported on TEVpE and TEVpH toxicity. Therefore we performed a viability test for expression of all three TEVp variants in  
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<p style="clear:left;">No data has previously been reported on TEVpE and TEVpH toxicity, therefore we expressed each TEVp variant in  
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HEK293T cells. Even after transfection with a high amount of the plasmid for each respective protease, the cells showed high viability, with practically
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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
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no difference when compared to control transfections (<ref>3</ref>).
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no difference when compared to cells transfected with a mock plasmid (<ref>3</ref>).
 
</p>
 
</p>
  
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<figcaption><b>Orthogonality and activity of TEVpE and TEVpH.</b><br/>   
 
<figcaption><b>Orthogonality and activity of TEVpE and TEVpH.</b><br/>   
 
<p style="text-align:justify">HEK293T cells were transfected with 2000ng of the indicated protease and 500ng of the indicated reporter. Cells
 
<p style="text-align:justify">HEK293T cells were transfected with 2000ng of the indicated protease and 500ng of the indicated reporter. Cells
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were lysed and analyzed by western blotting against the AU1 tag. The cleaved reporter (55 kDa) was detected only in the presence of the corresponding
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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.
 
TEVp variant.
 
</p>
 
</p>
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<figure data-ref="3">
 
<figure data-ref="3">
 
<img src="https://static.igem.org/mediawiki/2016/8/8f/T--Slovenia--4.4.3.png" >
 
<img src="https://static.igem.org/mediawiki/2016/8/8f/T--Slovenia--4.4.3.png" >
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<figcaption><b>Toxicity test of different TEVp variants for HEK203 cells.</b><br/>  
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<figcaption><b>Different TEVp variants are not toxic to HEK293T cells.</b><br/>  
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<p style="text-align:justify">Cells were transfected with plasmid DNA for different TEVp variants and counted two days later. Prior to counting
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<p style="text-align:justify">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.  
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cells were stained with trypan blue to determine the percentage of dead cells.
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</p>
 
</p>
 
</figcaption>
 
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<div>
 
<div>
 
<h3 style="clear:both"><span id = "hom" class="section">&nbsp;</span>TEVp homologs</h3>
 
<h3 style="clear:both"><span id = "hom" class="section">&nbsp;</span>TEVp homologs</h3>
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<p>Introduction of two new TEVp variants expanded our repertoire of tools, demonstrating that we can use the results of the mutational screening of
+
<p>Introduction of two new TEVp variants expanded our repertoire of tools, demonstrating that we can use the results of mutational screenings for
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protease variants, but a larger number of strictly orthogonal proteases would be required for modular design of logic gates. We therefore decided to  
+
protease variants. However, a larger number of strictly orthogonal proteases would be required for modular design of complex logic circuits. We therefore decided to  
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investigate activity of de novo created split proteases from the potyviridae family.
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investigate the activity of de novo created split proteases from the potyviridae family.
 
</p>
 
</p>
 
 
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</div>
 
</div>
 
<div class="content">
 
<div class="content">
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<p>The NIa proteases from the potyviridae group of plant viruses in general recognize a seven amino acid sequence motif as their substrate and
+
<p>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  
 
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  
 
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  
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</div><br/>
 
</div><br/>
 
 
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<p>We searched for sequences of different potyviruses available on UniProt, paying particular attention to any evidence of orthogonality among their target substrates.
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<p>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).
 
We decided to test the plum pox virus protease (PPVp), the soybean mosaic virus protease (SbMVp), and the sunflower mild mosaic virus protease (SuMMVp).
 
</p>
 
</p>
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<div class="content">
 
<div class="content">
 
<p>
 
<p>
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PPVp is one of the most studied potyviral proteases after the TEVp. Its substrate (PPVs) has an amino acid sequence NVVVHQ-A.
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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<x-ref>Zheng2008, Garcia1991</x-ref>.<br/>SbMVp has been  
 
In contrast to TEVp, it has been reported that PPVp is resistant to self-cleavage at the C-terminus<x-ref>Zheng2008, Garcia1991</x-ref>.<br/>SbMVp has been  
−
recently studied by Seo et al. as a tool for protein-protein interaction studies in the soybean. The substrate (SBMVs) has been determined to be the  
+
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 <x-ref>Seo2016, Yoon2000</x-ref>.<br>Similarly, SuMMVp has been used by Fernandez-Rodriguez et al.  
 
sequence ESVSLQ-S <x-ref>Seo2016, Yoon2000</x-ref>.<br>Similarly, SuMMVp has been used by Fernandez-Rodriguez et al.  
 
<x-ref>Fernandez-Rodriguez2016</x-ref>. in a study of genetic circuits using potyviral proteases. The substrate (SuMMVs) has been defined as the sequence  
 
<x-ref>Fernandez-Rodriguez2016</x-ref>. in a study of genetic circuits using potyviral proteases. The substrate (SuMMVs) has been defined as the sequence  
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</div><br/>
 
</div><br/>
 
 
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<p>All selected potyviral proteases were designed as synthetic genes and tested in mammalian cells for the activity using the
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<p>All selected potyviral proteases were designed as synthetic genes and their activity was tested in mammalian cells using
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<a href="https://2016.igem.org/Team:Slovenia/Protease_signaling/Reporters">cyclic luciferase reporters</a>, which results in the luciferase activity only upon cleavage.
+
<a href="https://2016.igem.org/Team:Slovenia/Protease_signaling/Reporters">cyclic luciferase reporters</a>, which propagates 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
 
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 (<ref>5</ref>).
 
their activity in the human cell chassis (<ref>5</ref>).

Revision as of 14:22, 18 October 2016

Orthogonality

Reporters Achievements Motivation 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.
  • We demonstrated higher cleavage activity of the TEVp homologues against their respective substrates in comparison to the already existing split TEVp.

 

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-toxicParks1994 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 (TEV(E)s) and TEVpH, cleaving the substrate ENLYFH-S (TEV(H)s). 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.

HEK293T cells were transfected with the indicated fLuc:TEVs and TEVp variant constructs. Luciferase activity was determined 24h 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).

Orthogonality and activity of TEVpE and TEVpH.

HEK293T cells were transfected with 2000ng of the indicated protease and 500ng 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 residueYoon2000, Nunn2005.


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).

Orthogonal proteases from the potyviridae family.

Homology models obtained from the crystal structure of TEVp (red) (PDB code: 1LVB) of PPVp (blue), SbMVp (cyan) and SuMMVp (yellow).

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-terminusZheng2008, 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 propagates 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.

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.