Part:BBa_K5477015
LexA-ERRγ Chimeric activator with LexA DNA binding domain fused with LBD of ERRγ
The LexA-ERRγ (LexA-Estrogen-Related Receptor Gamma) fusion protein combines the DNA-binding domain (DBD) of the LexA repressor with the ligand-binding domain (LBD) of Estrogen-Related Receptor Gamma (ERRγ), a member of the orphan nuclear receptor family. ERRγ is classified as an orphan receptor because, unlike classic nuclear receptors, it does not have a well-characterized endogenous ligand. Instead, it functions in a ligand-independent manner or is activated by synthetic or environmental ligands, allowing it to regulate a broad range of physiological processes, including energy metabolism, mitochondrial function, and cellular differentiation.
In the LexA-ERRγ fusion, the LexA DBD binds to LexA operator sequences (Lex6Op) in our reporter module, while the ERRγ LBD modulates transcriptional activation of NanoLuc.
The sequence for the LexA domain was from the paper of Zhou et al. 2022. This was fused with either the ligand-binding domain of the wild-type ERα, the mutant ERα or the Estrogen-Related Receptor gamma ERRγ. An alignment of the ligand-binding domains of the aforementioned was performed to determine the exact sequence to fuse with LexA to generate a chimerica activator that will bind to the Lex6Op in our reporter module.
Figure 1 - Multiple Sequence Alignment of Estrogen receptors - alpha and beta with Estrogen-related receptor gamma
Figure 2 - 3D Structure Prediction of Estrogen-related receptor gamma showing residue 217 found in the loop. The curved green line divides the structure and shows the ligand-binding domain of ERRγ. From residue 217 until the end of the amino acid sequence is fused downstream with LexA DNA binding domain to generate the chimeric activator.
In our system, this part was used as a receptor module to detect estrogenic compounds like BPA that have endocrine disrupting effects in breast milk (1) (2) (3) (4). For more details about how this part was used in our devices, visit the following links:
Results from Drylab
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 856
Illegal PstI site found at 1003
Illegal PstI site found at 1150 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 856
Illegal PstI site found at 1003
Illegal PstI site found at 1150 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 698
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 856
Illegal PstI site found at 1003
Illegal PstI site found at 1150 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 856
Illegal PstI site found at 1003
Illegal PstI site found at 1150
Illegal AgeI site found at 899 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 902
References
1. Ayami Matsushima, Takamasa Teramoto, Yoshimitsu Kakuta, Crystal structure of endocrine-disrupting chemical bisphenol A and estrogen-related receptor γ, The Journal of Biochemistry, Volume 171, Issue 1, January 2022, Pages 23–25, https://doi.org/10.1093/jb/mvab145
2. Çiftçi S, Yalçın SS, Samur G. Bisphenol A Exposure in Exclusively Breastfed Infants and Lactating Women: An Observational Cross-sectional Study. J Clin Res Pediatr Endocrinol. 2021 Nov 25;13(4):375-383. doi: 10.4274/jcrpe.galenos.2020.2021.0305. Epub 2021 Mar 22. PMID: 33749218; PMCID: PMC8638632.
3. Takayanagi S, Tokunaga T, Liu X, Okada H, Matsushima A, Shimohigashi Y. Endocrine disruptor bisphenol A strongly binds to human estrogen-related receptor gamma (ERRgamma) with high constitutive activity. Toxicol Lett. 2006;167(2):95-105. doi:10.1016/j.toxlet.2006.08.012
4. Zhou, T., Liang, Z. & Marchisio, M.A. Engineering a two-gene system to operate as a highly sensitive biosensor or a sharp switch upon induction with β-estradiol. Sci Rep 12, 21791 (2022). https://doi.org/10.1038/s41598-022-26195-x
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