Part:BBa_K2356000

CT33 with mCherry and Strep-tag II

Main info

The sequence is designed by TU-Eindhoven 2017[http://2017.igem.org/Team:TU-Eindhoven] and 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.

In short:

• 1018 DNA basepairs
• 334aa protein (36 kDa)
• Binds to Streptavidin
• Binds to 14-3-3
• Red fluorescent
• Flexible linkers

mCherry

In many biological or chemical processes it is convenient to allow visualization of the behavior of molecules. One facile approach to such visualization is the attachment of a fluorophore, such as mCherry. This red, monomeric protein is often used for this purpose and exhibits excitation and emission peaks at 587 and 610 nm, respectively.[1] Using this domain in a protein network, where other proteins comprise different fluorophores, may yield significant information on interactions and localization.

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.[2] 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.[3]

CT33

The 14-3-3 protein family is a well known group of dimeric proteins that are capable of binding multiple different molecules. 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.[4] The last 33 amino acids of this part are the same as the last 33 amino acids of H+ATPase, except the mutation of the last three to YDI, allowing unphosphorylated binding as well. Because this group is C-terminal, it has been named "CT33". It should noted that in other research, sometimes the last 52 amino acids are used, which is called "CT52". The domain is flanked by SalI and SacI restriction sites, allowing exchange of CT33 with CT52.

The binding of unphosphorylated CT33 and CT52 with YDI mutation to 14-3-3 family has extensively been researched and it was shown that this binding was particularly strong to the specific T14-3cΔC protein.[5] This happened in the presence of a small molecule, called fusicoccin, which functions as stabilizer and resulted in a Kd of 0.25 µM.[6]

Due to this low Kd value and tunability of fusicoccin this binding is interesting for contributing to a PPI network based on 14-3-3 scaffolds, especially when the valency of 14-3-3 can be altered.

Results

This part was successfully expressed in the pET28a(+) vector in E. coli BL21-DE3 cells upon addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) . One advantage of fluorophore addition is that the protein synthesis can be confirmed by measuring fluorescence. Besides the clearly visible red/purple color of the solution, the emission spectrum confirmed to presence of mCherry, with the characteristic peak aroun 610 nm, indicating that mCherry was correctly synthesized and folded.

Microscopy of interaction with 14-3-3 tetramer
An even better indication of close proximity between the two constructs can be generated by using a microscope. With the microscope we can make two pictures, one where we visualize GFP and one where we visualize mCherry, and overlay them to see that the two different constructs are close to each other. </br> Additionally, we can even visualize large clusters by measuring one of the fluorophores and compare this with all the other states described above.

In Figure_2">Figure 2, fluorescence microscopy images are shown after 48 h of incubation. As can be seen from the overlay spectra, the presence of fusicoccin as well as the presence of Strep-tactin is crucial for network formation. Without fusicoccin, no network formation is visible after 48 h. Without the presence of Strep-tactin, the mCherry and GFP signals are in close proximity, but it is unclear whether network formation has occured. This indicates that our system works as expected: a network is formed in presence of an inducer, and multivalency is also key to network formation.

" Figure 2: Fluorescence microscopy images after 48 h of incubation. Signal from samples with all GUPPI components (A, B and C) and samples without streptactin (D, E and F), leaving the CT33 protein with a single valency. A) GFP signal B) mCherry signal C) signal overlay D) GFP signal E) mCherry signal F) signal overlay. Data was modified using ImageJ.

References

[1] Shaner NC et al., Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nature Biotechnology 22, 2004:1567-1572. doi:10.1038/nbt1037
[2] 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
[3] https://www.iba-lifesciences.com/strep-tactin-system-technology.html
[4] Morsomme P, Boutry M. The plant plasma membrane H+-ATPase : structure , function and regulation. 2000;1465.
[5] 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.
[6] Hamer A Den, Lemmens LJM, Nijenhuis MAD, et al. Small-Molecule-Induced and Cooperative Enzyme Assembly on a 14-3-3 Scaffold. 2017:331-335. doi:10.1002/cbic.201600631.

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]