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 reconstituted ubiquitin. It is mainly comprised of the extracellular part of the human soluble interleukin-6 receptor and of the N-terminal part of split ubiquitin. Development of split-ubiquitin as a tool for study of protein-protein interactions in vivo, was first published in 1994 and has been an essential feature in biologists’ toolbox ever since [1]. A specific mutation in the N-terminal part protects it from binding spontaneously to the C-terminal part, however, reassociation can be facilitated by binding of a pair of proteins to which the split-ubiquitin parts are fused. Human interleukin-6 receptor was expressed in yeast for the first time in 1996 and further improvements paved the way to our own chimeric transmembrane proteins [2].

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 endoplasmatic 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.

Biosensor assays

References

[1] Johnsson, N., & Varshavsky, A. (1994). Split ubiquitin as a sensor of protein interactions in vivo. Proceedings of the National Academy of Sciences of the United States of America, 91(22), 10340–10344. https://doi.org/10.1073/pnas.91.22.10340 https://www.pnas.org/content/pnas/91/22/10340.full.pdf

[2] Vollmer, P., Oppmann, B., Voltz, N., Fischer, M., & Rose-John, S. (n.d.). 438±446 (1999) q FEBS 1999. In Eur. J. Biochem (Vol. 263). https://www.sciencedirect.com/science/article/abs/pii/S0022175996001639

[3] Chen, X., Zaro, J. L., & Shen, W. C. (2013). Fusion protein linkers: Property, design and functionality. In Advanced Drug Delivery Reviews (Vol. 65, Issue 10, pp. 1357–1369). https://doi.org/10.1016/j.addr.2012.09.039