Part:BBa_K1831002

The IceQueen T3-10 Scaffold

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,BBa_K1189011) [1] [2]. 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. The 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 complementary ‘E’ coil domain (such as our Type III AFPs: BBa_K1831001 [3], 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.

Scaffold Expression and Assembly

The expression of the scaffold unit itself was explored. Expression according to the Baker/Yeates protocol showed high protein expression rates and confirmed subunit size. The His-tag on the C-terminus was used to purify the protein using a Ni2+ column. The result of the purification steps is shown in Figure 1. E1, E2 and E3 were combined and used for determination of assembly size. For more information please visit our wiki page about our project; http://2015.igem.org/Team:Queens_Canada/AFP_Scaffold

Figure 1. Unmodified T3-10 SDS-PAGE gel.The entire protocol was performed to check expression, purification, and assembly of the scaffold.

The purified elution fractions were pooled and run through an S200 size exclusion column. The molecular weight of each subunit, calculated from the protein sequence9 is 22 kDa. The T3-10 scaffold is expected to assembly into a 12-mer unit with a minor portion as 9-mer units. Thus, for the S200 column used, the fully and partially assembled scaffolds are predicted to elute between 60-64 mL. The resultant chromatogram is shown in Figure 2 below. The results indicate that the majority of the protein is assembled in trimer state. Optimization of the assembly conditions is therefore required upon expression of the scaffold with the coil. This will be explored at a later time.

Figure 2. Unmodified T3-10 size exclusion chromatogram.

T3-10 Self-Assembling Scaffold with K-coil & His tag

Assembly Compatibility:

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

  Illegal BamHI site found at 728
  Illegal XhoI site found at 755
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]