Difference between revisions of "Part:BBa K1592002"
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[[File: HUST-China_2015_results_2.jpg|700px|thumb|center|Figure 1: Surface green fluorescence from anti si-tag-6xhis immunoassay was observed under 40X objective lens（Control is wildtype JMY1212 without plasmid. Test cell is the JMY1212 transformed with JMP62 plasmid. Regional enlargement shows a surface display of FITC labled Si-tag-6xhis protein）
 
[[File: HUST-China_2015_results_2.jpg|700px|thumb|center|Figure 1: Surface green fluorescence from anti si-tag-6xhis immunoassay was observed under 40X objective lens（Control is wildtype JMY1212 without plasmid. Test cell is the JMY1212 transformed with JMP62 plasmid. Regional enlargement shows a surface display of FITC labled Si-tag-6xhis protein）
 
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Figure 2 shows the result of our verification of cell surface display system. The fluorescence surrounding cell wall shows that we succeed in displaying the silica-tag protein onto the cell surface, which means YLcwp3 successfully anchor on the cell wall and display the interest protein outside of cell. For the limit of experimental conditions, we can not get a thorough fluorescence staining. Some cells can show a considerable flourescencent intensity, while some performs partial or weak flourescence which can not be detected by our microscope camera.  
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Figure 2 shows the result of our verification of cell surface display system. The fluorescence surrounding cell wall shows that we succeed in displaying the silica-tag protein onto the cell surface, which means YLcwp3 successfully anchor on the cell wall and display the interest protein outside of cell. Though due to the low resolution of our fluorescent microscope camera, we cannot show much clearer photos, but this result still successfully demonstrated the cell surface display of our silica binding proteins.
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Revision as of 14:47, 17 September 2015

Yarrowia lipolytica cell wall protein 3

The covalently bound GPI-anchored cell wall protein of Y. lipolytica Ylcwp1 has been isolated and characterized (Jaafar and Zueco 2004). The nucleotide sequence encoding 110 amino acids of the Ylcwp1 C terminus has been utilized for the construction of the cell surface display vector in Y. lipolytica (Yue et al. 2008). This system was successfully used to immobilize green fluorescent protein (Yue et al. 2008). The ability of five new putative covalently linked cell wall proteins of Y. lipolytica -Ylcwp1, Ylcwp2, Ylcwp3, Ylcwp4, Ylcwp5, was tested and the highest cell-bound lipase value was obtained with the anchor domains Ylcwp3 (Evgeniya et al. 2011), so we utilized the YLcwp3 for the cell surface display in Y. lipolytica.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 14
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 35

Characterization

As an anchor domain, YLcwp3 is the component of the cell surface display system of Yarrowia lipolytica.

Verification of cell surface display system

We used the fluorescence immunoassay to verify the success of cell surface display system. We had added the DNA sequence of 6xhis tag between the signal peptide and our silica-tag protein when constructing JMP62 plasmid, so that the 6xhis tag could be fusion expressed with the silica-tag protein and displayed on cell surface together. While the signal peptide could be cut out during the secretion. When we used the fluorescence immunoassay anti 6xhis tag, the primary antibody (mouse anti 6xhis tag) and the secondary antibody (FITC tagged goat anti-mouse IgG) detected 6xHis tagged Si-tag protein on cell surface.

Figure 1: Surface green fluorescence from anti si-tag-6xhis immunoassay was observed under 40X objective lens（Control is wildtype JMY1212 without plasmid. Test cell is the JMY1212 transformed with JMP62 plasmid. Regional enlargement shows a surface display of FITC labled Si-tag-6xhis protein）

Figure 2 shows the result of our verification of cell surface display system. The fluorescence surrounding cell wall shows that we succeed in displaying the silica-tag protein onto the cell surface, which means YLcwp3 successfully anchor on the cell wall and display the interest protein outside of cell. For the limit of experimental conditions, we can not get a thorough fluorescence staining. Some cells can show a considerable flourescencent intensity, while some performs partial or weak flourescence which can not be detected by our microscope camera.