Part:BBa_K5056004:Design
6X His-tag - de novo design anticancer peptide-12 - linker - cell-penetrating peptide
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 61
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 75
Illegal NgoMIV site found at 164 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 8
Design Notes
To effectively target the PDEδ, we first identify the 3D structure of it, which comprises 150 amino acids. Besides, we found the hotspot residues that were viable amino acids for our ACPs to bind within the PDEδ, specifically Y149, C56 and R61. This facilitates small molecules to bind with PDEδ. When our small molecules attach to PDEδ, the strong interactions cause changes in the shapes of the binding sites, in place of KRAS-PDEδ binding. By obtaining the structure of PDEδ from the Protein Data Bank(PDB), a database for 3D structure data of proteins and nucleic acids, we confirmed the structure of human PDEδ aligns with existing literature. The relevant code is 4JV6, which has specific structural features that make it a good target for protein design.
Two restriction sites (NdeI and SacI) have been added in both ends for digestion and ligation.
6X His tag has been added at the beginning for purification.
One restriction site, NheI, has been added between 6X His tag and the de novo anticancer peptide for future modification.
A middle linker is added between de novo anticancer peptides and cell-penetrating peptide
Polyarginine Cell-Penetrating Peptide: Peptides, especially intracellular functional peptides that can play a particular role inside a cell, have attracted attention as promising materials to control cell fate. However, hydrophilic materials like peptides are difficult for cells to internalize18. Octaarginine (R8) as a Polyarginine Cell-Penetrating Peptide (CPP) is reported to enhance peptide’s ability to cross the cell membrane and deliver cargo19. Hence, ‘RRRRRRRR’ is used as our signal peptide sequence for penetrating cell membrane and enter the cell.
Source
De novo anticancer peptide: The sequence was generated by RFdiffusion and ProteinMPNN-AI, so it is a all new peptide sequence. No source for this part.
Middle linker: Part:BBa_K243005
Cell-penetrating peptide: Hao, M., Zhang, L., & Chen, P. (2022). Membrane internalization mechanisms and design strategies of Arginine-Rich Cell-Penetrating peptides. International Journal of Molecular Sciences, 23(16), 9038. https://doi.org/10.3390/ijms23169038
References
Hao, M., Zhang, L., & Chen, P. (2022). Membrane internalization mechanisms and design strategies of Arginine-Rich Cell-Penetrating peptides. International Journal of Molecular Sciences, 23(16), 9038. https://doi.org/10.3390/ijms23169038