Protein_Domain

Part:BBa_K1478000

Designed by: Fabian Frömling   Group: iGEM14_Hannover   (2014-09-13)

Expa4 plant secretion signal, localizes to extracellular space

Coding region for first 20 aminoacids of plant protein Exansin4. Secretion signal for plants (derived from A. thaliana). When fused to a protein, directs the protein to extracellular space.

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
    COMPATIBLE WITH RFC[1000]


Functional Parameters

Bioinformatic analyses based on the raw protein sequence (20 aa) predicts the secretion of the protein with 50 % probability. For further analyses a GFP-fusion protein was designed. Therefore, Expa4 was cloned in frame with GFP and the cellulose binding domain. This coding sequence was cloned into a vector backbone and was used for plant transformation. The GFP signal was detected via confocal microscopy in plant cells. In large eucaryotic cells it’s possible to analyze the subcellular localization with fusion proteins. A prediction via bioinformatics showed nearly 100 % probability for the secretion of the fusion protein.

Figure 1 shows detected fluorescence of transformed Nicotiana tabacum cells. A GFP control without protein fusion is shown in lane A-D. The signal is specific for a cytosolic protein, which is indicated by chloroplast surrounded with GFP-Signal. In contrast the plasmamembranemarker (E-H) shows a specific signal at the periphery. The signal doesn’t surround the chloroplast, which shows that it is located at the cell cover. Because it’s a marker, it’s location is already known. The analyzed construct is shown in I-K. It contrast to cytoplasmic GFP, the signal appears to be in the exterior. The signal appears to diffuse between the cells, at the cell wall. This indicates that the secretion signal and the tethering at the cell wall via CBD might work.

Fig. 1: Confocal detection of fluorescence from transformed plant cells (replicate 1). Transformed cells of Nicotiana tabacum were analyzed via confocal microscopy. Column A-I: Red channel, shows chlorophyll’s autofluorescence which shows the chloroplasts. Column B-J: Green channel showing GFP. Column C-K: Red and green channel merged. Column D-L: Pseudo transmission detection. Lane A-D shows a transformed cell with raw GFP. Lane E-H shows a plasmamembranemarker (Nelson et al. 2007). Lane I-L shows the detection of the analyzed construct: Expa4-GFP-CBD.

[edit]
Categories
//chassis/eukaryote/athaliana
//chassis/eukaryote/ntabacum
//proteindomain/localization
Parameters
None