Difference between revisions of "Part:BBa K4260001"

Revision as of 15:57, 3 October 2022

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

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	[Link to wiki]

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.

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

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.

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

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]

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.

References

[1] TecCEM 2022

[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

@@ Line 29: / Line 29: @@
 ===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 Gly521, His524, Leu525 and Met528. It is also used as the biological receptor of some endocrine disrupting chemicals.
+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.