Part:BBa_K4260001:Design

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.

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.

Table I. Recopilation of the elements of the construct. [1]

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 - to create disulfide bonds with chitosan Added tags Histidine tag for Nickel Column