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          <b>Figure 1: </b>The presence of vesicular components modularly composed with coiled-coils and directly fused could be proven by western blotting. Top left) the exosomal marker cdc63 can be visualized with primary anti-cdc63 mouse-antibody and secondary anti-mouse antibody - horse radish peroxidase (HRP) fusion. CDC63 does not run as a tight band on the blot because of glycosylation patterns and its nature as a membrane protein. Top right) Gag-protein is determined at around 70 kDa. The fusion construct Gag-HiBiT-L7Ae shows some degradation corresponding to the molecular weight of L7Ae cleavage. Bottom) MCP RNA-binding proteins can be shown with anti-MCP antibodies.
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Revision as of 23:54, 21 October 2019


DHD154a

DHD154a is a double coiled-coil structure.

Usage

To increase the modularity of our system we decided to test coiled coil proteins. These coiled coils mimic the interactions of DNA helix-helix interactions. The coiled-coil protein interaction allows the loading of more than one protein and larger proteins. We are using these helical structures to load RNA binding proteins into exosomes or virus-like-particles.

Biology

We generated helical bundle heterodimers in which each monomer is a helix–turn–helix starting from four-helix backbones produced using a generalization of the Crick coiled-coil parameterization.[1]

Characterization

Western Blot

Figure 1: The presence of vesicular components modularly composed with coiled-coils and directly fused could be proven by western blotting. Top left) the exosomal marker cdc63 can be visualized with primary anti-cdc63 mouse-antibody and secondary anti-mouse antibody - horse radish peroxidase (HRP) fusion. CDC63 does not run as a tight band on the blot because of glycosylation patterns and its nature as a membrane protein. Top right) Gag-protein is determined at around 70 kDa. The fusion construct Gag-HiBiT-L7Ae shows some degradation corresponding to the molecular weight of L7Ae cleavage. Bottom) MCP RNA-binding proteins can be shown with anti-MCP antibodies.

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]


References

  1. ↑ Chen, Zibo ; Boyken, Scott E; Jia, Mengxuan ; Busch, Florian ; Flores-Solis, David ; Bick, Matthew J; Lu, Peilong ; VanAernum, Zachary L; Sahasrabuddhe, Aniruddha ; Langan, Robert A; Bermeo, Sherry ; Brunette, T J; Mulligan, Vikram Khipple ; Carter, Lauren P; DiMaio, Frank ; Sgourakis, Nikolaos G; Wysocki, Vicki H; Baker, David, Programmable design of orthogonal protein heterodimers Nature, 2018, ISSN: 1476-4687.