Difference between revisions of "Part:BBa K2926050"
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__NOTOC__ | __NOTOC__ | ||
<partinfo>BBa_K2926050 short</partinfo> | <partinfo>BBa_K2926050 short</partinfo> | ||
− | + | ||
<br> | <br> | ||
The N-teminal lectin binding domain of <i>S. cerevisiaes</i> protein Flo11 was N-terminally fused to the fluorescence reporter protein mCherry to enable protein visualisation. | The N-teminal lectin binding domain of <i>S. cerevisiaes</i> protein Flo11 was N-terminally fused to the fluorescence reporter protein mCherry to enable protein visualisation. | ||
+ | __TOC__ | ||
− | + | ==Usage and Biology== | |
− | To investigate processes of endocytosis we fused several <i/> S. cerevisiae</i> specific ligands as well as a short proline-glycine-peptide to mCherry. Those fusion proteins enable visualization of the ligand in- and outside the cell. Flo11 is a flocculation-mediating protein | + | <html> |
− | Flo_mCherry was characterized together with the three other fusion-proteins Mat_mCherry (<a href="https://parts.igem.org/Part:BBa_K2926049">BBa_K2926049</a>), Opy_mCherry (<a href="https://parts.igem.org/Part:BBa_K2926051">BBa_K2926051</a>) and Pro_mCherry (<a href="https://parts.igem.org/Part:BBa_K2926068">BBa_K2926068</a>) | + | To investigate processes of endocytosis we fused several <i/> S. cerevisiae</i> specific ligands as well as a short proline-glycine-peptide to mCherry. Those fusion proteins enable visualization of the ligand in- and outside the cell. Flo11 is a flocculation-mediating protein on the cell surface of <i>S. cerevisiae</i>. It has been shown that the N-terminal domain is responsible for homotypic binding (Douglas et al. 2007; Goossens und Willaert 2012; Karunanithi et al. 2010). |
+ | Flo_mCherry was characterized together with the three other fusion-proteins Mat_mCherry (<a href="https://parts.igem.org/Part:BBa_K2926049">BBa_K2926049</a>), Opy_mCherry (<a href="https://parts.igem.org/Part:BBa_K2926051">BBa_K2926051</a>) and Pro_mCherry (<a href="https://parts.igem.org/Part:BBa_K2926068">BBa_K2926068</a>). | ||
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</html> | </html> | ||
<partinfo>BBa_K2926050 SequenceAndFeatures</partinfo> | <partinfo>BBa_K2926050 SequenceAndFeatures</partinfo> | ||
− | |||
− | |||
+ | ==Protein purification== | ||
+ | <html> | ||
First, the marker protein mCherry (<a href="https://parts.igem.org/Part:BBa_J06504">BBa_J06504</a>) was cloned into the expression- and purification-vector pTXB1. | First, the marker protein mCherry (<a href="https://parts.igem.org/Part:BBa_J06504">BBa_J06504</a>) was cloned into the expression- and purification-vector pTXB1. | ||
To express the desired fusion-proteins the coding sequence of the specific ligands containing a short C-terminal | To express the desired fusion-proteins the coding sequence of the specific ligands containing a short C-terminal | ||
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with the theoretically determined spectra. | with the theoretically determined spectra. | ||
</div> | </div> | ||
− | + | </html> | |
− | < | + | ==Protein characterization== |
− | + | <html> | |
<div> | <div> | ||
A very important property of the fusion-proteins is the ability to fluoresce independently from the fusion at the | A very important property of the fusion-proteins is the ability to fluoresce independently from the fusion at the | ||
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fluorescence intensity of 0.54 µmol Texas Red. | fluorescence intensity of 0.54 µmol Texas Red. | ||
</div> | </div> | ||
+ | </html> | ||
+ | ==Endocytosis assays== | ||
− | |||
− | + | ===Fluorescence in the supernatant=== | |
− | + | <html> <div> | |
With the purified proteins we performed an endocytosis-assay (Fig. 10). <i>S. cerevisiae</i> was incubated over an hour with | With the purified proteins we performed an endocytosis-assay (Fig. 10). <i>S. cerevisiae</i> was incubated over an hour with | ||
1 µM fusion-protein. Every 15 minutes the fluorescence intensity in the supernatant was determined using a plate reader (Fig. 11). | 1 µM fusion-protein. Every 15 minutes the fluorescence intensity in the supernatant was determined using a plate reader (Fig. 11). | ||
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Our results indicate that it is possible to find target-specific ligands that selectively enhance endocytosis in the aimed cell while | Our results indicate that it is possible to find target-specific ligands that selectively enhance endocytosis in the aimed cell while | ||
other organisms do not even interact with them. | other organisms do not even interact with them. | ||
− | + | </html> </div> | |
− | < | + | ===Fluorescence microscopy=== |
+ | <html> | ||
<div> | <div> | ||
As a second proof that our ligands are specifically enhancing endocytosis in their target, we used fluorescence | As a second proof that our ligands are specifically enhancing endocytosis in their target, we used fluorescence | ||
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</div> | </div> | ||
<div> | <div> | ||
− | It could be observed that Mat_mCherry (upper right) and Opy_mCherry (lower | + | It could be observed that Mat_mCherry (upper right) and Opy_mCherry (lower left) were detectable within the cells. Mat_mCherry was taken up with a |
slightly higher efficiency than Opy_mCherry (data not shown). In contrast Flo_mCherry (lower right) seemed to form precipitates outside the cells while the | slightly higher efficiency than Opy_mCherry (data not shown). In contrast Flo_mCherry (lower right) seemed to form precipitates outside the cells while the | ||
negative control mCherry without any fusion has not been taken up by <i>S. cerevisiae</i>.<br> | negative control mCherry without any fusion has not been taken up by <i>S. cerevisiae</i>.<br> | ||
To conclude, we can say that our selected ligands mating factor alpha and the cysteine-rich domain of Opy2 as well as a short proline-peptide | To conclude, we can say that our selected ligands mating factor alpha and the cysteine-rich domain of Opy2 as well as a short proline-peptide | ||
− | were able to enhance endocytosis in the targeted cells. We also showed that Mat_mCherry is target-specific for <i>S. cerevisiae</i> so all in all | + | were able to enhance endocytosis in the targeted cells. We also showed that Mat_mCherry is target-specific for <i>S. cerevisiae</i> so all in all were able to proof our concept. It is possible to enter selected target cells via cell-specific ligands. |
− | + | </html> | |
+ | ==References== | ||
+ | <html> | ||
+ | Douglas, Lois M.; Li, Li; Yang, Yang; Dranginis, A. M. (2007): Expression and characterization of the flocculin Flo11/Muc1, a Saccharomyces cerevisiae mannoprotein with homotypic properties of adhesion. In: Eukaryotic cell 6 (12).<br> | ||
+ | |||
+ | Goossens, Katty V. Y.; Willaert, Ronnie G. (2012): The N-terminal domain of the Flo11 protein from Saccharomyces cerevisiae is an adhesin without mannose-binding activity. In: FEMS yeast research 12 (1).<br> | ||
+ | Karunanithi, Sheelarani; Vadaie, Nadia; Chavel, Colin A.; Birkaya, Barbara; Joshi, Jyoti; Grell, Laura; Cullen, Paul J. (2010): Shedding of the mucin-like flocculin Flo11p reveals a new aspect of fungal adhesion regulation. In: Current biology : CB 20 (15). | ||
</div> | </div> | ||
Latest revision as of 19:18, 18 October 2019
Fusion protein of Flo11 from yeast and mCherry
The N-teminal lectin binding domain of S. cerevisiaes protein Flo11 was N-terminally fused to the fluorescence reporter protein mCherry to enable protein visualisation.
Contents
Usage and Biology
To investigate processes of endocytosis we fused several S. cerevisiae specific ligands as well as a short proline-glycine-peptide to mCherry. Those fusion proteins enable visualization of the ligand in- and outside the cell. Flo11 is a flocculation-mediating protein on the cell surface of S. cerevisiae. It has been shown that the N-terminal domain is responsible for homotypic binding (Douglas et al. 2007; Goossens und Willaert 2012; Karunanithi et al. 2010).
Flo_mCherry was characterized together with the three other fusion-proteins Mat_mCherry (BBa_K2926049), Opy_mCherry (BBa_K2926051) and Pro_mCherry (BBa_K2926068).
Sequence and Features
Sequence was validated by Sanger sequencing.
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Protein purification
First, the marker protein mCherry (BBa_J06504) was cloned into the expression- and purification-vector pTXB1. To express the desired fusion-proteins the coding sequence of the specific ligands containing a short C-terminal glycine-serine-linker was successfully cloned into the vector pTXB1 coding for mCherry which resulted in four different pTXB1-constructs coding for the fusion-proteins Mat_mCherry, Flo_mCherry and Opy_mCherry. Those fusion-proteins were expressed in E. coli ER2566. The expression was easily detectable, as it was indicated by the red colour of the culture (Fig. 1 and 2).
Protein characterization
Endocytosis assays
Fluorescence in the supernatant
The same assay performed for S. cerevisiae was carried out for A. niger to verify the uptake of Pro_mCherry into the cells. Additionally, to investigate the specificity of the tested ligands, A. niger was also incubated with the S. cerevisiae-specific Mat_mCherry (Fig. 12).
Our results indicate that it is possible to find target-specific ligands that selectively enhance endocytosis in the aimed cell while other organisms do not even interact with them. </div>
Fluorescence microscopy
To conclude, we can say that our selected ligands mating factor alpha and the cysteine-rich domain of Opy2 as well as a short proline-peptide were able to enhance endocytosis in the targeted cells. We also showed that Mat_mCherry is target-specific for S. cerevisiae so all in all were able to proof our concept. It is possible to enter selected target cells via cell-specific ligands.
References
Douglas, Lois M.; Li, Li; Yang, Yang; Dranginis, A. M. (2007): Expression and characterization of the flocculin Flo11/Muc1, a Saccharomyces cerevisiae mannoprotein with homotypic properties of adhesion. In: Eukaryotic cell 6 (12).
Goossens, Katty V. Y.; Willaert, Ronnie G. (2012): The N-terminal domain of the Flo11 protein from Saccharomyces cerevisiae is an adhesin without mannose-binding activity. In: FEMS yeast research 12 (1).
Karunanithi, Sheelarani; Vadaie, Nadia; Chavel, Colin A.; Birkaya, Barbara; Joshi, Jyoti; Grell, Laura; Cullen, Paul J. (2010): Shedding of the mucin-like flocculin Flo11p reveals a new aspect of fungal adhesion regulation. In: Current biology : CB 20 (15).