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| − | Design
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| − | 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.
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| − | 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.
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| − | Figure 1. Construct sequence design.
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| − | Usage and Biology
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| − | Human Estrogen Receptor alpha is a 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 (NCBI Gene ID: 2099). 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.
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| − | 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 (Klein et al., 2014). 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.
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| − | Last but not least, OmpA signal peptide was retrieved from (Goulas et al., 2014) because of its efficiency.
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