Difference between revisions of "Part:BBa K1965024"
 
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<h3>Introduction </h3>
 
<h3>Introduction </h3>
  
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     TEVp and other potyviral proteases have been previously shown to be functional in mammalian cells, however Cesaratto
 
     TEVp and other potyviral proteases have been previously shown to be functional in mammalian cells, however Cesaratto
 
     et al. have shown that wild type TEV protease is not active in the lumen of the ER
 
     et al. have shown that wild type TEV protease is not active in the lumen of the ER
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     <x-ref>Cesaratto2015</x-ref>
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     <sup>[1]</sup>
 
     . They designed a TEV protease variant active in the endoplasmic reticulum by preventing two major types of
 
     . They designed a TEV protease variant active in the endoplasmic reticulum by preventing two major types of
 
     post-translational modifications: N-glycosylation and cysteine oxidation. To avoid these inhibiting modifications,
 
     post-translational modifications: N-glycosylation and cysteine oxidation. To avoid these inhibiting modifications,
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     KDEL at the C-terminus of the protein. In addition to these mutations, the C-terminal residues SELVYSQ (236–242)
 
     KDEL at the C-terminus of the protein. In addition to these mutations, the C-terminal residues SELVYSQ (236–242)
 
     were substituted with NEGGGLE, which blocks protease auto-cleavage
 
     were substituted with NEGGGLE, which blocks protease auto-cleavage
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     <sup>[1]</sup>
 
     .
 
     .
 
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<h3>Characterization</h3>
 
<h3>Characterization</h3>
  
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<p>In order to test erTEV function, we designed an ER localized reporter construct SS:TagRFP:AU1:TEVs:KDEL (link) with
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<p>In order to test erTEV function, we designed an ER localized reporter construct SS:TagRFP:AU1:TEVs:KDEL (<a href="https://parts.igem.org/Part:BBa_K1965028">BBa_K1965028</a>) with
 
     introduced TEVp recognition motif with aminoacid sequence ENLYFQ-S before the KDEL sequence on the C-terminal end of
 
     introduced TEVp recognition motif with aminoacid sequence ENLYFQ-S before the KDEL sequence on the C-terminal end of
 
     the reporter. Furthermore, this system was aimed to be used for inducible secretion from the lumen of ER by
 
     the reporter. Furthermore, this system was aimed to be used for inducible secretion from the lumen of ER by
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<h3 id="ref-title">References</h3>
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<h3>References</h3>
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<sup>[1]</sup>Cesaratto, F., López-Requena, A., Burrone, O. R., & Petris, G. (2015). Engineered tobacco
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etch virus (TEV) protease active in the secretory pathway of mammalian cells. Journal of Biotechnology, 212, 159–166.
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https://doi.org/10.1016/j.jbiotec.2015.08.026 <br>
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 16:55, 18 October 2016


erTEVp

Introduction

Endoplasmic reticulum active TEV (erTEVp) is a modified version of the 27kDa tobacco etches virus protease (TEVp). TEVp and other potyviral proteases have been previously shown to be functional in mammalian cells, however Cesaratto et al. have shown that wild type TEV protease is not active in the lumen of the ER [1] . They designed a TEV protease variant active in the endoplasmic reticulum by preventing two major types of post-translational modifications: N-glycosylation and cysteine oxidation. To avoid these inhibiting modifications, mutations N23Q, C130S and N171T were made. To ensure correct localization and accumulation of this TEVp variant inside the endoplasmic reticulum, we also attached a signal sequence (MDMRVLAQLLGLLLLCFPGARC) at the N-terminus and KDEL at the C-terminus of the protein. In addition to these mutations, the C-terminal residues SELVYSQ (236–242) were substituted with NEGGGLE, which blocks protease auto-cleavage [1] .

Characterization

In order to test erTEV function, we designed an ER localized reporter construct SS:TagRFP:AU1:TEVs:KDEL (BBa_K1965028) with introduced TEVp recognition motif with aminoacid sequence ENLYFQ-S before the KDEL sequence on the C-terminal end of the reporter. Furthermore, this system was aimed to be used for inducible secretion from the lumen of ER by triggered removal of the KDEL sequence.

This construct was expressed under the CMV promoter. The coding sequence for erTEVp was deposited in pSB1C3.

When erTEVp was present and active in the ER, the KDEL sequence of the reporter was removed from the reporter and the protein was secreted from the cell, which we detected with western blot ( 1 ).

Cleavage with erTEV facilitates secretion of reporter from ER lumen.
The reporter was detected in the medium of cells only when cotransfected with erTEVp. HEK293T cells were transfected with the indicated constructs. Reporters were detected with WB in the concentrated medium.

References

[1]Cesaratto, F., López-Requena, A., Burrone, O. R., & Petris, G. (2015). Engineered tobacco etch virus (TEV) protease active in the secretory pathway of mammalian cells. Journal of Biotechnology, 212, 159–166. https://doi.org/10.1016/j.jbiotec.2015.08.026


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 784