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.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 856
  Illegal PstI site found at 1003
  Illegal PstI site found at 1150
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 856
  Illegal PstI site found at 1003
  Illegal PstI site found at 1150
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 698
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 856
  Illegal PstI site found at 1003
  Illegal PstI site found at 1150
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 856
  Illegal PstI site found at 1003
  Illegal PstI site found at 1150
  
• 1000
  COMPATIBLE WITH RFC[1000]