Part:BBa_K2356000

CT33 with mCherry and Strep-tag II

Main info

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 should be able to be used to bind 14-3-3 protein scaffolds to tetrameric Streptavidin proteins.

Strep-tag II

The middle part of the sequence encodes for the so called "Strep-tag II", consisting of the peptide sequence WSHPQFEK. This sequence has proven to exhibit a high binding affinity towards streptavidin.[3] This binding can be utilized for multiple purposes, which is why this short peptide sequence is so essential. At first the binding to streptavidin can be utilized in the formation of large Protein-Protein Interaction (PPI) networks, due to the tetrameric structure of streptavidin. The creation of such large network could have many different purposes, such as gelation and/or phase separation. Meanwhile, the Strep-tag II sequence is also extensively used in protein purification purposes. The Strep-tag II is able to selectively bind to columns containing Strep-tactin, a variant of streptavidin engineering by IBA Life Sciences. Since the tag is so small, it does not interfere with the folding of the protein.[4]

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.

The protein mass 36 kDa.

References

[3] Schmidt GM, Skerra A, The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins. Nature protocols 2007;2(6):1528-35. doi:10.1038/nprot.2007.209
[4] https://www.iba-lifesciences.com/strep-tactin-system-technology.html
[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]