BBa_K1831000

NpuDnaE intein circularization construct using Type III AFP and a computer-optimized linker

What: Starting from the NpuDnaE intein RFC [105] circularization construct BioBrick from Team Heidelberg 2014 (BBa_K1362000) we developed a computationally-optimized linker sequence to circularize a Type III antifreeze protein. Our linker consists of the tripeptide GAA, which in addition to the extein scar CWE/RGK, links the N and C termini of a the Type III AFP (PDB file: 1AME) without straining the core protein structure or distorting the residues critical for ice-binding. Our BioBrick also added a constitutive T7 promoter (Part BBa_I14018) allowing expression of your circular protein without any additional sub-cloning steps.

Why: Type III AFPs have a variety of potential applications in the oil and gas, food, and health industries, however these industries often use processes involving extreme temperatures, pH levels, and salt concentrations. Type III AFP is a particularly fragile protein as it denatures at 37^oC. Circularizing the protein using our BioBrick affords greater stability at a wider variety of experimental conditions

BBa_K1831001

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 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). There is 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, see below). 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.

BBa_K1831002

T3-10 Scaffold with K-coiled-coil + His tag

What: A single subunit of the T3-10 self-assembling protein scaffold (Baker 2008) is fused in-frame to the K-coil from Team Calgary 2013’s parallel coiled-coil proteins (BBa_K1189010). The 'K' denotes the charged lysine residues in the ‘e’ and ‘g’ positions of the helical wheel representation of the coils. A His-tag fused to the C-terminus of the coils allows quick purification. A flexible linker Gly-Ser between the K-coil and the His-tag prevents potential electrostatic interference between the two domains.

Why: This fusion of the K-coil to each individual subunit means that once assembled into a multimer, multiple proteins with a complimentary E-coil domain (such as our Type III AFPs: BBa_K1831001, BBa_K1831003) can be non-covalently attached to the scaffold. By adding a flexible linker of Gly-Ser we incorporated a unique BamHI restriction site. This acts as a diagnostic marker for simplified cloning of our BioBrick into the expression vector of your choosing.

BBa_K1831003

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). 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), and not the AFP with E-coil, we have enabled simple separation of any non-interacting coiled-coil domains.