Part:BBa_K1831001
IceQueen Antifreeze Protein with E-coil & His-tag
Type III AFP with E-coil + His tag
What: This BioBrick is a fusion protein consisting of the E-coil domain of our coiled-coil system fused in-frame to the C-terminus of a Type III AFP. Type III AFP (PDB fIle: 1AME) is a moderately active antifreeze protein from the ocean pout, with a small (7Kb), globular structure. The name 'E-coil' refers to the coiled-coil with glutamic acid residues in the ‘g’ [1] and ‘e’ [2] positions of the helical wheel first used by Team Calgary 2013 in the coiled-coil linkers BBa_K1189010 and BBa_K1189011. The 'K' and 'E' coils are then only able to form heterodimeric complexes, creating a strong, yet highly specific means of associating two different proteins. In our part, we included a His-tag at the C-terminus of the E-coil which provides an alternative purification method to ice-affinity.
Why: The fusion of the E-coil to the Type III AFP will enable non-covalent, yet high affinity, specific attachment of AFPs to our protein scaffold and K-coil BioBrick BBa_K1831002 [3]. The interaction between the ‘E’ and ‘K’ coils involved oppositely charged residues, promoting the heterodimeric interaction of the coils that links AFPs to our scaffold. The addition of a His-tag to the C-terminus of the coil will allow for simple purification of the protein even for teams without access to an ice-affinity purification apparatus.
TypeIII and AFP E-coil fusion + His tag
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 331
Illegal XhoI site found at 358 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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