Part:BBa_K1763421:Design

PCquad Mutant 10

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 408
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 105
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 768
  

Design Notes

Considered the area to insert the cleavage sequence. This mutation was selected by examining the secondary structure to ensure minimal disruption of existing alpha-helices or beta sheets, sites were sandwiched by glycine residues, and used PyMOL to check if the site would be accessible to the protease. Geometric accessibility by the protease thrombin and minimal speculated extent of conformational changes by insertion.

Using previous crystallographic data, we visualized the protein cage using pymol (Figure 2). We then identified potential insertion sites based on two criteria:

1. The insertion had to be sterically accessible to protease binding

2. The insertion had to theoretically have minimal effects on initial protein cage assembly.

We successfully cloned our top protein cage mutant (Mutant 10), based on the above criteria.

Source

Bromoperoxidase (trimer) and M1 Virus Matrix Protein (dimer) linked with an alpha helix. Ligated onto BBa_K525998 to add a T7 promoter and RBS.

References

Lai, Y.-T., Cascio, D. & Yeates, T. O. Structure of a 16-nm Cage Designed by Using Protein Oligomers. Science (80-. ). 336, 1129–1129 (2012).

Lai, Y.-T. & Al, E. Structure and Flexibility of Nanoscale Protein Cages Designed by Symmetric Self-Assembly. J Am Chem Soc 135, 7738–7743 (2013).