Difference between revisions of "Part:BBa K2356003"
| Line 14: | Line 14: | ||
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. | 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 is 35 kDa. | ||
Revision as of 10:15, 27 October 2017
CT33 with Strep-tag II and mCherry
The sequence starts with DNA coding for Strep-tag II, allowing it to bind to Strep-Tactin or other Streptavidin variants. This is followed by DNA encoding for mCherry, a fluorophore. 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 in pET28a(+) and led to the creation of the desired protein. This protein can, for example, 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 (PPIs), giving it two purposes at once.
About 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 is 35 kDa.
[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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 49
Illegal BamHI site found at 769 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 793
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 20