Part:BBa_K3809010:Design

ESR1 with Linker, His Tag and Signal Peptide

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 48
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1264
  

Design Notes

The idea of the desing of this part was that if we could express this protein receptor, purify it and immobilize it, we could potentially use it to capture Endocrine Disruptive Compounds in a sample of water. In order to check if EDCs could bind to the receptor, we used molecular docking with the estrogen receptor obtained from PDB (code: 2IOG) and different chemicals that include estradiol, BPA, phenol, benzene and different pthalates. The result we obtained was adequate, since the binding constants of all the EDCs were similar or better to the binding constant of the natural ligand of the receptor (estradiol). We figured that we needed to design a way to purify and immobilize the protein after its expression. We therefore added a Signal Peptide Sequence to guarantee its secretion from E. coli; a His Tag to purify it using a Ni Affinity Column; and a linker sequence so that there would not be any problems in the folding of both domains. The linker sequence has an Cystein that could help us immobilize our protein in a functionilized surface. To verify that the receptor could fold with all the changes we made, we used a model prediction software and checked if there werer any structural changes compared to the original protein. Therefore, we made a structure alignment between our model and protein 2IOG from PDB which was the original sequence we used in the first place. We also added two M13 primer sites for its amplification. They are located before the Prefix and After the Suffix.

In order to express the protein in a bacterial system, we optimized the codons for E. coli using Benchling.

Source

The Signal Peptide sequence NPS4 was obtained from BBa_K3606042, the linker was a modified version of BBa_K1486004, with the addition of a cistein; ESR1 sequence was obtained from Homo sapiens estrogen receptor 1 (ESR1), transcript variant 1, mRNA, NCBI Reference Sequence: NM_000125.4.

References

References: [1] Y. Song et al., “High-resolution comparative modeling with RosettaCM”, Structure, vol 21, no 10, bll 1735–1742, Sep 2013. [2] K. D. Dykstra et al., “Estrogen receptor ligands. Part 16: 2-Aryl indoles as highly subtype selective ligands for ERα”, Bioorganic & Medicinal Chemistry Letters, vol 17, no 8, bll 2322–2328, 2007. [3] E. F. Pettersen et al., “UCSF Chimera--a visualization system for exploratory research and analysis”, J Comput Chem, vol 25, no 13, bll 1605–1612, Oct 2004. [4] C. J. Williams et al., “MolProbity: More and better reference data for improved all-atom structure validation”, Protein Sci, vol 27, no 1, bll 293–315, Nov 2017. [5] S. Kim et al., “PubChem in 2021: new data content and improved web interfaces”, Nucleic Acids Res, vol 49, no D1, bll D1388–D1395, Jan 2021. [6] A. Grosdidier, V. Zoete, en O. Michielin, “SwissDock, a protein-small molecule docking web service based on EADock DSS”, Nucleic Acids Res, vol 39, no Web Server issue, bll W270-7, May 2011.