Coding
ESR1_HD

Part:BBa_K4260001

Designed by: Brenda Jimenez, Karen Marin, Melchor Soto   Group: iGEM22_TecCEM   (2022-09-23)
Revision as of 19:06, 8 October 2022 by AmielRosete (Talk | contribs)


ESR1: Estrogen Receptor 1 with periplasmic signal peptide OmpA, GGGGSC linker and histidine tag


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE 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), this is a receptor protein which aim is to bind to estrogens. This protein keeps important amino acids sites where different ligands bind. It is also used as the biological receptor of some endocrine disrupting chemicals.

Therefore, we used the genomic coding sequence of Homo sapiens Estrogen Receptor 1 (ESR1) optimizing its codons for an E.coli expression. We added a (GGGGSC) linker, this one is composed of four glycines, one serine and one cysteine, with the purpose of attaching hER alpha protein to chitosan, thus, ensuring the desired position of the molecule showing the estrogen binding site up for an effective capture. The periplasmic signal peptide element “OmpA” helps the cellular machinery to speed up the process of protein expression and send it to the periplasmic space, where it can be purified using the histidine tag for a nickel column. Figure 1 illustrates the detailed design of this part.


T--TecCEM--registry-ESR1proteincoding-Design1.jpeg

Figure 1. Construct sequence design.


Sources, usage and biology

Fig.2:Protein ESR1 complexed to estradiol. PDB 1a52 for visualization only. Taken from 10.2210/pdb1A52/pdb

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

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). 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] [5]

Fig.3:Periplasmic cell space. For visualization only. Created in BioRender

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].

Histidine tag

Base Pairs: 18 bp

Histidine tag was chosen for an easy and standardized purification using a Nickel Affinity Column chromatography.

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.

Methodology

We 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)



Fig. 4 Molecular simulation of our BBa_K4260001 including ESR1 coding sequence (green) and CSGGGG linker (purple).
Protein designed by TecCEM 2022. modeled at Chimera.

Molecular docking between hER alpha and BisphenolA:
Fig 5. BPA molecule, PubChem (6623) and
visualized at Chimera.
Fig 6. Interactions between hER alpha and ligand
BPA in residues Gly406, Lys407, Phe410, Leu416,
Asp417; interactions given by ProteinPlus - Pose
view and modeled at Chimera.
Fig 7. BPA molecule, PubChem (6623) and
visualized at Chimera.


Molecular docking between hER alpha and Carbamazepine:
Fig 8. Carbamazepine molecule, PubChem (2554)
and visualized at Chimera.
Fig 9. Interactions between hER alpha and ligand carbamazepine in residues Gly406, Phe410, Leu414; interactions given by ProteinPlus - Pose view and modeled at Chimera.
Fig 10. Molecular docking of our BBa_K4260001
with one of its ligands CBZ (Orange); including
ESR1 coding sequence (green) and CSGGGG
linker (purple). Protein designed by TecCEM 2022.
Modeled by AutoDock Vina at Chimera.


Molecular docking between hER alpha and Estradiol:
Fig 11. Estradiol molecule, the natural ligand for
hER alpha, PubChem (5757) and visualized
at Chimera.
Fig 12. Interactions between hER alpha and
ligand Estradiol in residues Trp399,Ser474,
Leu472,Lys478; interactions given by
ProteinPlus - Pose view and modeled
at Chimera.
Fig 13. Molecular docking of our BBa_K4260001
with one of its ligands Estradiol (blue); including
ESR1 coding sequence (green) and CSGGGG
linker (purple). Protein designed by TecCEM 2022. Modeled by AutoDock Vina at Chimera.

References

[1] TecCEM 2022 https://2022.igem.wiki/teccem/

[2] NCBI Gene ID: 2099 https://www.ncbi.nlm.nih.gov/gene/2099

[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] TecCEM 2021 https://2021.igem.org/Team:TecCEM

[6] 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

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