Part:BBa_K3617004

sIL-6R-nTEV

This biobrick is a part of a 2-protein system that is designed for detection of human interleukin-6 and transduction of the signal by means of a reconstitution of a split TEV protease. The split TEV was originally developed by Wehr, M. C. et al. In 2006[[#References[1]]] for measuring protein-protein interaction. This part resembles that of sIL-6R-Nub in which the extrecellular receptor remains the human interleukin-6 receptor, but now with a split TEV protease, an N-terminal part (amino acid residues 1-118) and a C-terminal part (amino acid residues 119-242), fused with a flexible linker on the C-terminal of the receptorbound transmembrane domain (TMD). Thus, binding of the interleukin to the receptor results in a reconstitution of the two halfes nTEV and cTEV, thus rendering it active. The activated protease will cleave a recognition site on a flexible linker bound to another TMD, which releases the synthetic transciption factor LexA-VP16.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 130
  Illegal BglII site found at 502
  Illegal XhoI site found at 456
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

This biobrick consists of multiple parts; An endoplasmic reticulum import signal peptide from the Saccharomyces cerevisiae cell wall integrity and stress response component 1 (Wsc1) receptor in S. cerevisiae , the second and third domain of human soluble interleukin-6 receptor subunit alpha (sIL-6R), the transmembrane receptor of Wsc1 and the N-terminal part of the split version of ubiquitin, constituting the first 34 amino acids of ubiquitin. The domain possesses the Ile13Gly mutation which inhibits the spontaneous association of the two split protein halves by reducing their affinity to each other. Between the sIL-6R domains and the transmembrane domain, a flexible 2XXGGGGS linker [3] exists. Between the transmembrane domain and the N-terminal split ubiquitin domain two basic amino acids (KR) have been added together with the 2XGGGGS linker.

Sequence optimization

The sequence was codon optimize for S. cerevisiae. The recognition sequences for SpeI, XbaI, NotI, EcoRI, PstI were avoided to follow the RFC10 standard.

Biosensor assays

References

[1] Wehr, M. C., Laage, R., Bolz, U., Fischer, T. M., Grünewald, S., Scheek, S., Bach, A., Nave, K. A., & Rossner, M. J. (2006). Monitoring regulated protein-protein interactions using split TEV. Nature Methods, 3(12), 985–993. https://doi.org/10.1038/nmeth967

[2]

[3]