Part:BBa_K2356000

CT33 with mCherry and Strep-tag II

The sequence starts with DNA coding for mCherry, a fluorophore. This is followed by DNA coding for Strep-tag II, allowing it to bind to Strep-Tactin or other Streptavidin variants. The last part of the sequence encodes for CT33, a protein domain comprising the final 33 amino acids of the C-terminus of H+-ATPase, a known binding partner of 14-3-3 scaffolds. The parts are connected via linkers, consisting mostly of Glycine and Serine. Expression of the part was succesful and led to the creation of the desired protein. This protein can be used to bind 14-3-3 protein scaffolds. to tetrameric Streptavidin proteins.

Strep-tag II
The binding of Strep-tag II to Streptavidin is suitable for protein purification purposes, but this binding may also be utilized in protein-protein interactions, giving it two purposes at once.

CT33
One motif that is known to bind to 14-3-3 is the phosphorylated C-terminus of H+-ATPase, an enzyme that catalyzes the hydrolysis of ATP to ADP.[1] In this project we use peptides compromising the final 33 and 52 amino acids of this C-terminus, which is referred to as CT33.. In previous research the binding of unphosphorylated CT52 (comprising the final 52 amino acids of H+-ATPase instead of the last 33) to T14-3cΔC was established by mutation of the last three amino acids of CT52 to YDI and addition of fusicoccin, yielding a Kd of 0.85 nM.[2] Due to this low value and tunability of fusicoccin this binding is interesting for contributing to a PPI network based on 14-3-3 scaffolds. The CT33 DNA sequence can be exchanged for a CT52 sequence by making use of the flanking SalI and SacI restriction sites.

[1] Morsomme P, Boutry M. The plant plasma membrane H ‡ -ATPase : structure , function and regulation. 2000;1465.
[2] Ottmann C, Marco S, Jaspert N, et al. Article Structure of a 14-3-3 Coordinated Hexamer of the Plant Plasma Membrane H + -ATPase by Combining X-Ray Crystallography and Electron Cryomicroscopy. 2007:427-440. doi:10.1016/j.molcel.2006.12.017.

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 22
  Illegal BamHI site found at 751
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 772
  
• 1000
  COMPATIBLE WITH RFC[1000]