Difference between revisions of "Part:BBa K1965028"

 
 
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<partinfo>BBa_K1965028 short</partinfo>
 
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<body><h3>Introduction</h3>
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<p>TagRFP is a fluorescent protein used as a reporter protein for visualization with confocal microscopy and for FRET. Merzlyak et al.<sup>[2]</sup> developed it by modifying the wild type RFP from the sea anemone <i>Entacmaea quadricolor</i> to prolong its fluorescence lifetime and make it less susceptible to pH <sup>[2]</sup>. With the addition of the AU1 tag, we engineered the reporter to also be detected with western blot.</p>
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<p>We used this construct as a reporter for ER retention and release. The IgG kappa signal sequence <a href=”http://www.uniprot.org/uniprot/P01601”>(UNIPROT: P01601)</a> fused to its N-terminus enables cotranslational translocation into endoplasmic reticulum (ER). The C-terminal KDEL sequence then enables interaction with the KDEL receptor on the ER membrane and thereby retention of reporter in the ER lumen (or more accurately retrieval of the reporter from cis Golgi apparatus back to ER) <sup>[3,4]</sup>.</p>
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<p>We inserted a Tobacco etch virus protease (TEVp) cleavage site (TEVs) before the KDEL retrieval signal, to allow for induced secretion. Cleavage by a variant of TEVp active in the ER<sup>[1]</sup>(<a href="https://parts.igem.org/Part:BBa_K1965024">BBa_K1965024</a>) was designed to remove the KDEL sequence, thereby targeting it to secretion. </p>
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<p>When the SS:TagRFP:AU1:TEVS:KDEL reporter (TagRFP<sup>KDEL</sup>) (<ref>1</ref>A) was expressed from a CMV promoter without an active erTEVp we confirmed its localization in the ER with confocal microscopy (<ref>1</ref>B). Additionally, we could not detect any TagRFP in the cell medium with Western blotting, proving the construct is effectively retained in the ER. When erTEVp was present and active in the ER, the KDEL sequence was removed from the reporter and the protein was secreted from the cell, which we detected with Western blot (<ref>1</ref>C), demonstrating that proteolytic activity in the ER can regulate protein secretion. </p>
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<p>The secreted TagRFP was detected as a slightly larger protein than expected, most probably due to N-glycosylation of the reporter in the secretory pathway. With our other TagRFP-based reporter (<a href=" https://static.igem.org/mediawiki/2016/b/b4/T--Slovenia--6.2.3.png "> SS:TagRFP:AU1:FurS:TM:TEVS:KKMP </a>) we showed that incubation of the secreted TagRFP with N-glycosidase F resulted in a band of the expected size.</p>
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          <figcaption><b> Cleavage with ER-redesigned protease (erTEV) facilitates secretion of reporter from cells. </b><br/>(A) Scheme of the reporter with cleavable KDEL retrieval signal and protease target motif.
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(B) The reporter with the KDEL retrieval signal was localized in the ER. HEK293T cells were transfected with the indicated reporters and in (C) also with TEVp. Localization was detected with confocal microscopy. (C) 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.  </figcaption>
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</div>
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<h3>References</h3>
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<sup>[1]</sup>Cesaratto, F., López-Requena, A., Burrone, O. R. & Petris, G. Engineered tobacco etch virus (TEV) protease active in the secretory pathway of mammalian cells. J. Biotechnol. 212, 159–66 (2015).<br>
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<sup>[2]</sup>Merzlyak, E. M. et al. Bright monomeric red fluorescent protein with an extended fluorescence lifetime. Nat. Methods 4, 555–557 (2007). <br>
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<sup>[3]</sup>Munro, S. & Pelham, H. R. A C-terminal signal prevents secretion of luminal ER proteins. Cell 48, 899–907 (1987). <br>
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<sup>[4]</sup>Stornaiuolo, M. et al. KDEL and KKXX retrieval signals appended to the same reporter protein determine different trafficking between endoplasmic reticulum, intermediate compartment, and Golgi complex. Mol. Biol. Cell 14, 889–902 (2003). <br>
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Latest revision as of 17:29, 18 October 2016


ss-TagRFP-AU1-tevS-KDEL

Introduction

TagRFP is a fluorescent protein used as a reporter protein for visualization with confocal microscopy and for FRET. Merzlyak et al.[2] developed it by modifying the wild type RFP from the sea anemone Entacmaea quadricolor to prolong its fluorescence lifetime and make it less susceptible to pH [2]. With the addition of the AU1 tag, we engineered the reporter to also be detected with western blot.

We used this construct as a reporter for ER retention and release. The IgG kappa signal sequence (UNIPROT: P01601) fused to its N-terminus enables cotranslational translocation into endoplasmic reticulum (ER). The C-terminal KDEL sequence then enables interaction with the KDEL receptor on the ER membrane and thereby retention of reporter in the ER lumen (or more accurately retrieval of the reporter from cis Golgi apparatus back to ER) [3,4].

We inserted a Tobacco etch virus protease (TEVp) cleavage site (TEVs) before the KDEL retrieval signal, to allow for induced secretion. Cleavage by a variant of TEVp active in the ER[1](BBa_K1965024) was designed to remove the KDEL sequence, thereby targeting it to secretion.

When the SS:TagRFP:AU1:TEVS:KDEL reporter (TagRFPKDEL) (1A) was expressed from a CMV promoter without an active erTEVp we confirmed its localization in the ER with confocal microscopy (1B). Additionally, we could not detect any TagRFP in the cell medium with Western blotting, proving the construct is effectively retained in the ER. When erTEVp was present and active in the ER, the KDEL sequence was removed from the reporter and the protein was secreted from the cell, which we detected with Western blot (1C), demonstrating that proteolytic activity in the ER can regulate protein secretion.

The secreted TagRFP was detected as a slightly larger protein than expected, most probably due to N-glycosylation of the reporter in the secretory pathway. With our other TagRFP-based reporter ( SS:TagRFP:AU1:FurS:TM:TEVS:KKMP ) we showed that incubation of the secreted TagRFP with N-glycosidase F resulted in a band of the expected size.

Cleavage with ER-redesigned protease (erTEV) facilitates secretion of reporter from cells.
(A) Scheme of the reporter with cleavable KDEL retrieval signal and protease target motif. (B) The reporter with the KDEL retrieval signal was localized in the ER. HEK293T cells were transfected with the indicated reporters and in (C) also with TEVp. Localization was detected with confocal microscopy. (C) 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. Engineered tobacco etch virus (TEV) protease active in the secretory pathway of mammalian cells. J. Biotechnol. 212, 159–66 (2015).
[2]Merzlyak, E. M. et al. Bright monomeric red fluorescent protein with an extended fluorescence lifetime. Nat. Methods 4, 555–557 (2007).
[3]Munro, S. & Pelham, H. R. A C-terminal signal prevents secretion of luminal ER proteins. Cell 48, 899–907 (1987).
[4]Stornaiuolo, M. et al. KDEL and KKXX retrieval signals appended to the same reporter protein determine different trafficking between endoplasmic reticulum, intermediate compartment, and Golgi complex. Mol. Biol. Cell 14, 889–902 (2003).


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.rc site found at 697
    Illegal SapI.rc site found at 79