Part:BBa_K5477013

LexA-ERα - Chimeric activator with LexA DNA binding domain fused with ERα-LBD

The LexA-ERα (LexA-Estrogen Receptor Alpha) fusion protein combines the DNA-binding domain of the bacterial LexA repressor with the ligand-binding domain (LBD) of the Estrogen Receptor alpha (ERα). This engineered protein is designed to utilize the ligand-binding specificity of ERα and the DNA-binding specificity of the LexA operator system. In this fusion, the LexA DBD binds to LexA operator sequences (Lex6Op), while the ERα LBD allows the regulation of gene expression in response to estrogen or estrogen-like molecules, in this case BPA. When BPA binds to the ERα LBD within this fusion protein, it induces a conformational change that activates our reporter module.

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 chimeric activator that will bind to the Lex6Op in our reporter module.

<img src="" alt="Multiple Sequence Alignment" width="500" height="auto">

Figure 1 - Multiple Sequence Alignment of Estrogen receptors - alpha and beta with Estrogen-related receptor gamma (CLUSTALW)

<img src="" alt="3D Structure of Estrogen Receptor" width="500" height="auto">

Figure 2 - 3D Structure Prediction of Estrogen receptor alpha showing residue 281 found in the loop. The curved green line divides the structure and shows the ligand-binding domain of ERα. From residue 281 until the end of the amino acid sequence is fused downstream with LexA DNA binding domain to generate the chimeric activator.

Usage and Biology

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 900
  Illegal PstI site found at 1084
  Illegal PstI site found at 1255
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1084
  Illegal PstI site found at 1255
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1034
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 900
  Illegal PstI site found at 1084
  Illegal PstI site found at 1255
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 900
  Illegal PstI site found at 1084
  Illegal PstI site found at 1255
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

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