Difference between revisions of "Part:BBa K1965030"
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TagRFP is a fluorescent protein used as a reporter protein for visualization with confocal microscopy and for FRET. Merzlyak et al. 1 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. With the addition of the AU1 tag, we engineered the reporter to also be detected with western blot.
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We used this construct as a reporter for ER retention and release. The IgG kappa signal sequence (UNIPROT: P01601) fused to its N-terminus of TagRFP  and the transmembrane sequence (TM, BBa_ K157010) enables cotranslational translocation into the membrane of the endoplasmic reticulum (ER). The C-terminal KKMP sequence then enables interaction with the KKMP receptor on the ER membrane and thereby retention of reporter in the ER (or more accurately retrieval of the reporter from cis Golgi apparatus back to ER).
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We inserted three copies of a Tobacco etch virus protease (TEVp) cleavage site (TEVS) before the KKMP retrieval signal, to allow for induced secretion. Cleavage by a variant of TEVp active in the ER4 was designed to remove the KDEL sequence, thereby targeting it to secretion. The addition of the furin cleavage site (furS) between the TagRFP and the transmembrane domain ensures cleavage of the reporter from the membrane in the trans Golgi apparatus. This allowed us to design our constructs so that they are cleaved off of the membrane without any modified scar sequences attached to them.
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Localization of the ss:TagRFP:AU1:furS:TM:3xtevS:KKMP (TagRFP:TM:KKMP) reporter was confirmed by the confocal microscopy. We used a control reporter without the KKMP retention signal (TagRFP:TM) which we detected both on the ER and the plasma membrane (Figure 3.A). In case of the present KKMP retention signal, the reporter was detected only on the ER (Figure 3.B). When TagRFP:TMKKMP was coexpressed with TEVp, localization of the reporter was similar to the localization of the positive control (TagRFP:TM) on the plasma membrane and the ER (Figure 3.C).
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<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>[1]</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. With the addition of the AU1 tag, we engineered the reporter to also be detected with western blot.
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<p>We used this construct as a reporter for ER retention and release. The IgG kappa signal sequence (UNIPROT: P01601) fused to N-terminus of TagRFP and the transmembrane sequence (<a href=" https://parts.igem.org/Part:BBa_K157010 "> BBa_ K157010 </a>) enable cotranslational translocation into the membrane of the endoplasmic reticulum (ER). The C-terminal KKMP sequence then enables interaction with the KKMP receptor on the ER membrane and thereby retention of reporter in the ER (or more accurately retrieval of the reporter from cis Golgi apparatus back to ER). <sup>[2,3]</sup>.</p>
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<p>We inserted three copies of a Tobacco etch virus protease (TEVp) cleavage site (TEVS) before the KKMP retention signal, to allow for induced secretion. The addition of the furin cleavage site (furS) <sup>[4]</sup> between the TagRFP and the transmembrane domain ensures cleavage of the reporter from the membrane in the trans Golgi apparatus. This allowed us to design our constructs so they are cleaved off the membrane without any modified scar sequences attached to them.</p>
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<h3>Characterization</h3>
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<p>Localization of SS:TagRFP:AU:furS:TM:3xtevS:KKMP (TagRFP:TM<sup>KKMP</sup>) reporter was confirmed by confocal microscopy. We used a control reporter without the KKMP retention signal (TagRFP:TM) which we detected both on the ER and the plasma membrane (<ref>1A</ref>). In case of the present KKMP retention signal, the reporter was detected only on the ER (<ref>1B</ref>). When TagRFP:TM<sup>KKMP</sup> was coexpressed with TEVp, localization of the reporter was similar to the localization of the positive control (TagRFP:TM)( <a href="https://parts.igem.org/Part:BBa_K1965029">BBa_K1965029</a>) on the plasma membrane and the ER (<ref>1C</ref>).</p>
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<p>A band with a slightly larger apparent size than the expected size of TagRFP (28 kDa) was detected by western blotting in cells transfected with TagRFP:TM. We showed that the unexpected difference in size was due to glycosylation, as we detected the protein at the expected size after deglycosilation of the sample with N-glycosidase F. We were unable to detect a corresponding band in the medium of cells transfected with TagRFP:TM<sup>KKMP</sup> in the absence of the protease.</p>
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<p>Together, these results confirm that localization and secretion of the protein reporter with the transmembrane domain depends on the presence and proteolysis of the KKMP retention signal and that proteolysis can be used to induce secretion of already synthesized protein.</p>
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    <figure data-ref="1">
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        <img class="ui medium image" src=" https://static.igem.org/mediawiki/2016/b/b4/T--Slovenia--6.2.3.png ">
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        <figcaption><b> Localization of protease-responsive reporters on ER membrane depending on the proteolysis.</b><br/>(A) The transmembrane reporter without the KKMP retention signal was localized both on the ER and plasma membrane. (B) The transmembrane reporter with the KKMP retention signal was localized exclusively on the ER membrane. (C) After cleavage of the KKMP retention signal, the transmembrane reporter translocated to the plasma membrane. HEK293T cells were transfected with the indicated reporters and in (C) also with TEVp. Localization was detected with confocal microscopy. Each image is accompanied with a scheme of the transfected construct. (D) Glycosylated reporter was detected in the medium of cells transfected with the transmembrane reporter without the KKMP retention signal. HEK293T cells were transfected with the indicated constructs. Reporters were detected with WB in the concentrated medium. In lane 2, sample was incubated with N-glycosidase F.
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<h3>References</h3>
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<sup>[1]</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>[2]</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>[3]</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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<sup>[4]</sup>Tian, S., Huang, Q., Fang, Y. & Wu, J. FurinDB: A Database of 20-Residue Furin Cleavage Site Motifs, Substrates and Their Associated Drugs. Int. J. Mol. Sci 12, 1060–1065 (2011). <br>
  
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A band with a slightly larger apparent size than the expected size of TagRFP (28 kDa) was detected by western blotting in cells transfected with TagRFP:TM:KKMP. We showed that the unexpected difference in size was due to glycosylation, as we detected the protein at the expected size after deglycosilation of the medium sample with N-glycosidase F. We were unable to detect a corresponding band in the medium of cells transfected with TagRFP:TM:KKMP in the absence of the protease.
 
  
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Together, these results confirm that localization and secretion of the protein reporter with the transmembrane domain depends on the presence and proteolysis of the KKMP retention signal and that proteolysis can be used to induce secretion of already synthesized protein.
 
  
  

Revision as of 16:54, 18 October 2016


ss:TagRFP:AU1:furS:TM:3xtevS:KKMP

Introduction

TagRFP is a fluorescent protein used as a reporter protein for visualization with confocal microscopy and for FRET. Merzlyak et al. [1] 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. 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 N-terminus of TagRFP and the transmembrane sequence ( BBa_ K157010 ) enable cotranslational translocation into the membrane of the endoplasmic reticulum (ER). The C-terminal KKMP sequence then enables interaction with the KKMP receptor on the ER membrane and thereby retention of reporter in the ER (or more accurately retrieval of the reporter from cis Golgi apparatus back to ER). [2,3].

We inserted three copies of a Tobacco etch virus protease (TEVp) cleavage site (TEVS) before the KKMP retention signal, to allow for induced secretion. The addition of the furin cleavage site (furS) [4] between the TagRFP and the transmembrane domain ensures cleavage of the reporter from the membrane in the trans Golgi apparatus. This allowed us to design our constructs so they are cleaved off the membrane without any modified scar sequences attached to them.

Characterization

Localization of SS:TagRFP:AU:furS:TM:3xtevS:KKMP (TagRFP:TMKKMP) reporter was confirmed by confocal microscopy. We used a control reporter without the KKMP retention signal (TagRFP:TM) which we detected both on the ER and the plasma membrane (1A). In case of the present KKMP retention signal, the reporter was detected only on the ER (1B). When TagRFP:TMKKMP was coexpressed with TEVp, localization of the reporter was similar to the localization of the positive control (TagRFP:TM)( BBa_K1965029) on the plasma membrane and the ER (1C).

A band with a slightly larger apparent size than the expected size of TagRFP (28 kDa) was detected by western blotting in cells transfected with TagRFP:TM. We showed that the unexpected difference in size was due to glycosylation, as we detected the protein at the expected size after deglycosilation of the sample with N-glycosidase F. We were unable to detect a corresponding band in the medium of cells transfected with TagRFP:TMKKMP in the absence of the protease.

Together, these results confirm that localization and secretion of the protein reporter with the transmembrane domain depends on the presence and proteolysis of the KKMP retention signal and that proteolysis can be used to induce secretion of already synthesized protein.

Localization of protease-responsive reporters on ER membrane depending on the proteolysis.
(A) The transmembrane reporter without the KKMP retention signal was localized both on the ER and plasma membrane. (B) The transmembrane reporter with the KKMP retention signal was localized exclusively on the ER membrane. (C) After cleavage of the KKMP retention signal, the transmembrane reporter translocated to the plasma membrane. HEK293T cells were transfected with the indicated reporters and in (C) also with TEVp. Localization was detected with confocal microscopy. Each image is accompanied with a scheme of the transfected construct. (D) Glycosylated reporter was detected in the medium of cells transfected with the transmembrane reporter without the KKMP retention signal. HEK293T cells were transfected with the indicated constructs. Reporters were detected with WB in the concentrated medium. In lane 2, sample was incubated with N-glycosidase F.

References

[1]Merzlyak, E. M. et al. Bright monomeric red fluorescent protein with an extended fluorescence lifetime. Nat. Methods 4, 555–557 (2007).
[2]Munro, S. & Pelham, H. R. A C-terminal signal prevents secretion of luminal ER proteins. Cell 48, 899–907 (1987).
[3]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).
[4]Tian, S., Huang, Q., Fang, Y. & Wu, J. FurinDB: A Database of 20-Residue Furin Cleavage Site Motifs, Substrates and Their Associated Drugs. Int. J. Mol. Sci 12, 1060–1065 (2011).
Sequence and Features BBa_K1965030 SequenceAndFeatures