Part:BBa_K1831003

IceQueen Antifreeze Protein with E-coil

Type III AFP with E-coil

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 E-coil refers to the coiled-coil with glutamic acid residues in the ‘e’ and ‘g’ positions of the helical wheel first used by Team Calgary 2013 (BBa_K1189011). This protein has been shown to be purified effectively by ice-affinity. If a His-tag is required for your purposes, see (BBa_K1831001).

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 [1]). 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. There is no His-tag, but ice-affinity purification can be used. By including a His-tag on our K-coil fusion protein (our scaffold: BBa_K1831002 [2]), and not this AFP with E-coil, we have enabled simple separation of any non-interacting coiled-coil domains. If you require a His-tag on your protein you may use our non-tagged version: BBa_K1831001 [3]

The modified Type III AFP was expressed and purified using ice affinity purification. This technique uses the ability of AFPs to bind to an ice shell to separate the protein from other lysate components. The exploitation of this property enables simultaneous purification and activity confirmation of an antifreeze protein.

Two rounds of ice affinity purification were performed with the AFP – E coil construct with the liquid fraction representing the components not incorporated into the ice shell and the ice fraction the thawed ice shell from each round. The results of these rounds are shown in Figure 1. Round 1 saw the incorporation of number of non-specific components. However, the second round of purification saw the desired protein as the predominant inclusion and provided sufficient purification for experimental purposes. Figure 2 demonstrates the size difference between the AFP-E coil and the wild-type AFP.

Figure 1. Ice affinity purification of AFP-E coil.The supernatant represents the pre-shell supernatant lysate from protein expression. The first round of ice affinity purification included the incorporation of a number of components into the ice fraction. The second round of purification left the predominant component as the AFP with the attached E coil.

Figure 2. Visualization of the slight difference in size between the wild-type AFP and the AFP-Ecoil.

Type III AFP with E-coil

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 331
  Illegal XhoI site found at 343
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]