Difference between revisions of "Part:BBa K197027"
Line 4: Line 4:
 
This is a spacer used between the displayer and passenger for e coli cell surface display.  The beta roll domain is frequently observed in secreted proteins.  This part comes from alkaline protease of Pseudomonas aeruginosa.  The crystal structure of the part shows that the beta roll domain contains a repetitive all-beta structure that binds calcium ions in its turns.  The binding of calcium, whose concentration is higher in the extracellular environment than in the cytoplasm, may act as a switch that prevents the premature folding of proteins in the periplasm or cytoplasm of host cells.  We have observed that including this part can improve the display of proteins on the surface of E. coli.
 
This is a spacer used between the displayer and passenger for e coli cell surface display.  The beta roll domain is frequently observed in secreted proteins.  This part comes from alkaline protease of Pseudomonas aeruginosa.  The crystal structure of the part shows that the beta roll domain contains a repetitive all-beta structure that binds calcium ions in its turns.  The binding of calcium, whose concentration is higher in the extracellular environment than in the cytoplasm, may act as a switch that prevents the premature folding of proteins in the periplasm or cytoplasm of host cells.  We have observed that including this part can improve the display of proteins on the surface of E. coli.
  
−
{{Berkeleywetlab2009}}
+
{{Berkeleywetlab2009_spacer}}
  
  

Revision as of 04:51, 22 October 2009

Beta Roll

This is a spacer used between the displayer and passenger for e coli cell surface display. The beta roll domain is frequently observed in secreted proteins. This part comes from alkaline protease of Pseudomonas aeruginosa. The crystal structure of the part shows that the beta roll domain contains a repetitive all-beta structure that binds calcium ions in its turns. The binding of calcium, whose concentration is higher in the extracellular environment than in the cytoplasm, may act as a switch that prevents the premature folding of proteins in the periplasm or cytoplasm of host cells. We have observed that including this part can improve the display of proteins on the surface of E. coli.


This part is a cell-surface displayer spacer part. A spacer is used to improve the display of passenger domains.

For successful cell surface display of proteins, there must be an effective protein localization mechanism. Gram-negative bacteria such as E. Coli have two membranes, which present a problem for transporting proteins synthesized in the cytoplasm to the outside of the cell. Various transport schemes exist in gram-negative bacteria to effectively localize proteins to the outermembrane. The most common schemes are TypeI, TypeIII, and TypeV secretion.

HeatMapBerkeley.jpg

The heat map above points to an interesting trend made clear by the streptavidin and mgfp-5 data. Although all constructs contain short linkers between the displayers and passengers, the inclusion of spacer elements for both systems appears to enhance functional surface display of the passengers. Moreover, the identity of the spacer element is an important parameter determining display efficiency. There is an increase in functional display when the INP repeats spacer is added between the displayers and the strep tag as is seen in the increase in lighter blocks in the map. This trend is especially evident in the mgfp data in which there are several weak signals (many dark blocks in the map) for mgfp displayed on its own. With the addition of several spacer elements, a significant general increase in signal for almost 100% of the systems is observed.


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]