Part:BBa_K4260001:Design
ESR1: Estrogen Receptor 1 with periplasmic signal peptide OmpA, GGGGSC linker and histidine tag
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| ESR1_HD shortlist resume | |
|---|---|
| Function | Periplasmic expression of Human
Estrogen Receptor Alpha protein |
| Optimization for | E.coli strains |
| Signal Peptide | OmpA-periplasmic expression |
| Linker | GGGGSC - create disulfide bonds
with chitosan |
| Added tags | Histidine tag for Nickel Column |
| Submitted by | Hydro-Defense TecCEM 2022 [1] |
Design
The TecCEM team 2022 designed this sequence for the codification of the Human Estrogen Receptor Alpha (hERa). A receptor protein which aim is to bind estrogens and that has affinity to Endocrine Disrupting Chemicals due to important amino acids [2]. Therefore, we used the genomic coding sequence of Homo sapiens Estrogen Receptor 1 (ESR1) optimizing its codons and added features for expression. Thus, a linker composed of four glycines, one serine and one cysteine [3][4] was added as well with the purpose of attaching hER alpha to chitosan and ensure a desired position of the molecule showing the estrogen binding sites up for an effective capture. The signal peptide element helps the cellular machinery to speed up the process of protein expression and send it to the periplasmic space [5][6] , where it can be purified using the histidine tag for a nickel affinity column [6]. Table 1 and figure 1 gives the detailed design of this part.
Figure 1. Construct sequence design.
Sources, usage and biology
Coded protein
Name: Estrogen Receptor Alpha
Origin: Homo sapiens
Synonyms:ER; ESR; Era; ESRA; ESTRR; NR3A1
Base Pairs: 2111 bp
CDS:coding sequence from nucleotide 232 to 2019 of mRNA from NM_000125.4 isoform 1. [2]
Gene type: protein coding (P03372-UniProt)
Properties:It's affinity to estrogens, estradiol, and endocrine disrupting chemicals.
Nuclear transcription factor whose biological duty is to regulate cellular signaling to enhance physiological processes in humans, in the body it needs hER beta to create a functional complex. For the matter of the project, only the hER alpha is going to be described. ESR1 comes from genomical Homo sapiens ESR1. It contains the elements for coding a protein including its N-terminal ligand transactivation domain, DNA binding domain, hinge domain and the C- terminal ligand transactivation domain (retrieved from NCBI)[8]. hER alphas role is to keep on going the regulation of transcriptional genes inducible by estrogens, thus, enhancing cellular signaling corresponding to metabolic, endocrine, nervous, reproductive systems between others.
Linker
Base Pairs: 18 bp
Linkers are short amino acid sequences that act as spacers between protein domains within a protein. The ones containing Glycines are flexible, separating domains and mostly, creating covalent bonds between proteins. Adding Serine as a polar residue reduces linker protein interaction preserving protein function [3]. Finally, the last residue being cysteine was added to create a disulfide bond with chitosan for surface immobilization, thus keeping the strategy developed by TecCEM 2021 [4][8]
Omp A
Base Pairs: 63 bp
Last but not least, OmpA (Outer membrane protein) signal peptide was retrieved from literature because of its efficiency as periplasmic expression signal peptide [5][6].
Histidine tag
Base Pairs: 18 bp
Histidine tag was chosen for an easy and standardized purification using a Nickel Affinity Column chromatography.[7]
Characterization: protein modeling and molecular docking
Objective
Observe molecular interactions between Human Estrogen Receptor Alpha hER alpha_HD22 coded by BBa_K4260001 and some of its ligands reported in literature such as Estradiol (natural ligand), Carbamazepine, Bisphenol A and Diethyl Phthalate, chemical molecules that acts as Endocrine Disruptors [9]
MethodologyWe first modeled our protein sequence hER alpha_HD22 through I-TASSER and the given results were modeled at Chimera, the same as the ligands downloaded from PubChem. We executed the docking hER alpha-ligands using AutoDock Vina and each result was submitted to Protein Plus to observe the interactions between ligands and the protein. Then, returning to the docking, we located these given amino acids to verify if the union matched. The results are shown below.
Protein Model of our designed receptor molecule: Human Estrogen Receptor Alpha (hERα_HD22)
| Molecular docking between hER alpha and BisphenolA: | ||
|---|---|---|
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visualized at Chimera. |
BPA in residues Gly406, Lys407, Phe410, Leu416, Asp417; interactions given by ProteinPlus - Pose view and modeled at Chimera. |
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| Molecular docking between hER alpha and Carbamazepine: | ||
|---|---|---|
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and visualized at Chimera. |
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| Molecular docking between hER alpha and Estradiol: | ||
|---|---|---|
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hER alpha, PubChem (5757) and visualized at Chimera. |
ligand Estradiol in residues Trp399,Ser474, Leu472,Lys478; interactions given by ProteinPlus - Pose view and modeled at Chimera. |
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| Molecular docking between hER alpha and Diethyl phthalate: | ||
|---|---|---|
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ligand Diethyl phthalate in residues Tyr136; interactions given by ProteinPlus - Pose view and modeled at Chimera. |
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Molecular docking of our designed receptor molecule (hERα_HD22) with all the ligands before mentioned:
Conclusion
Our protein keeps essential amino acids and regions where ligands such as Bisphenol A, Carbamazepine, Diethyl phthalate and Estradiol have a great affinity. The interactions of these ligands with the amino acids indicated by Protein Plus are observed and confirmed by docking.
Also, in this modeling we observed that a wide range of the ligands' possible interaction residues are not close to the linker and most of them are in the opposite site, leaving that space for the immobilization on chitosan and accommodating the protein as we expect.
References
[1] TecCEM 2022 https://2022.igem.wiki/teccem/ [2] Paterni, I., Granchi, C., Katzenellenbogen, J. A., & Minutolo, F. (2014). Estrogen receptors alpha (ERα) and beta (ERβ): subtype-selective ligands and clinical potential. Steroids, 90, 13–29. https://doi.org/10.1016/j.steroids.2014.06.012
[3] Joshua S. Klein, Siduo Jiang, Rachel P. Galimidi, Jennifer R. Keeffe, Pamela J. Bjorkman. (2014) Design and characterization of structured protein linkers with differing flexibilities. Protein Engineering, Design and Selection, Volume 27, Issue 10, Pages 325–330. https://doi.org/10.1093/protein/gzu043
[4] Chen, X., Zaro, J. L., & Shen, W.-C. (2013). Fusion protein linkers: Property, design and functionality. Advanced Drug Delivery Reviews, 65(10), 1357–1369. doi:10.1016/j.addr.2012.09.039
[5] Goulas T, Cuppari A, Garcia-Castellanos R, Snipas S, Glockshuber R, Arolas JL, et al. (2014) The pCri System: A Vector Collection for Recombinant Protein Expression and Purification. PLoS ONE 9(11): e112643.https://doi.org/10.1371/journal.pone.0112643
[6] https://parts.igem.org/Part:BBa_K208003
[7] IBA Solutions for life sciences. (2021, August). Expression and purification of proteins using 6xHistidine-tag. IBA. https://www.iba-lifesciences.com/media/a8/ee/aa/1631860506/Manual-6xHistidine-tag.pdf
[8] Paterni, I., Granchi, C., Katzenellenbogen, J. A., & Minutolo, F. (2014). Estrogen receptors alpha (ERα) and beta (ERβ): subtype-selective ligands and clinical potential. Steroids, 90, 13–29. https://doi.org/10.1016/j.steroids.2014.06.012 [8] iGEM TecCEM 2021. [9] Ronderos-Lara, J. G. , Saldarriaga-Noreña, H., Reyes-Romero, P. G. , Chávez-Almazán, L. A. , Vergara-Sánchez, J., Murillo-Tovar, M. A. , & Torres-Segundo, C. (2020). Emerging Compounds in Mexico: Challenges for Their Identification and Elimination in Wastewater. In (Ed.), Emerging Contaminants. IntechOpen. https://doi.org/10.5772/intechopen.93909