Part:BBa_K3113068

DHD154a

DHD154a is a heterodimeric 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."

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 CD63 can be visualized with primary anti-CD63 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]