Part:BBa_K3617000

sIL-6R-Nub

This biobrick is an ORF encoding a fusion protein consisting of :

• The first 21 amino acids (Signal peptide for import to endoplasmatic reticulum) of the endogenous Cell wall integrity and stress response component 1 (Wsc1 [Jon: the protein is usually refered to as Wsc1 while the gene is called SLG1 what do we call it?) receptor in S. Cerevisiae.
• The second and third domain (aa 115-365) of human soluble interleukin-6 receptor subunit alpha (sIL-6R).
• The transmembrane domain of Wsc1
• N-terminal part of a split version of ubiquitin. This means the first 34 amino acids of the ubiquitin protein. Compared to Wt ubiquitin, the domain has a mutation(Ile13Gly). This mutation inhibits the spontaneous association of the two split protein halves by reducing the affinity.
• Between the sIL-6R domains and the transmembrane domain we added a flexible 2XGGGGS linker (cite: PMID: 23026637) and between the transmembrane domain and the n-terminal split ubiquitin domain we added two basic amino acids; KR, and the 2XGGGGS linker again.

Expected function of the protein: The signal peptide and transmembrane domain constitute the backbone of our modular framework for localizing our receptors at the plasma membrane as type I single pass transmembrane proteins. As a type I transmembrane protein the soluble interleukin receptor domains would be localized extracellularly while the N-terminal part of the split protein would be the intracellular. Ivanusic et al. (citation) introduced the use of the signal peptide and transmembrane domain in a split-ubiquitin system for screening for PPIs at the plasma membrane in S. cerevisiae. The two fibronectin type III soluble interleukin-6 receptor subunit alpha domains are - as seen in crystal structures of the receptor mediating the binding of the receptor to interleukin-6 (see fig. 1). The outer Ig-like domain of the receptor mediates other functions of the receptor (vollmer et al. PMID: 10406952) and it is OMITTED in this part - this might cause the unwanted localization as addressed later! This biobrick is intended to work together with (biobrick) which has the outer three domains of the IL-6 co-receptor soluble glycoprotein 130 (sgp130), extracellularly and the C-terminal part of split ubiquitin intracellularly with the synthetic transcription factor linked to the C-terminal of the split ubiquitin domain. We hypothesized that BBa_K3617000 (this biobrick) and (andet biobrick nummer) would both localize to the same membrane but that they would be dissociated in the absence of interleukin-6. In the presence of interleukin-6, we imagined that the extracellular domains of the two parts; IL-6R and sgp130, would associate into a heterotrimer consisting of IL-6, IL-6R and sgp130. The trimerization would cause intracellular complementation of the ubiquitin that can then be recognized by an endogenous deubiquitinizing enzyme which releases the transcription factor resulting in expression of a reporter. Unfortunately, our assays indicated that the biobricks do not work together as intended.

The sequence was codon optimized for S. cerevisiae, subsequently the sequence was modified by interchanging synonymous codons in the signal peptide region and in the flexible linkers and transmembrane domain to make the part fit into our modular framework where we can easily interchange intra- and extracellular domains while avoiding too long identical sequences which might cause unwanted homologous recombination. Furthermore we avoided following recognition sequences SpeI, XbaI, NotI, EcoRI, PstI to both follow the RFC10 standard and make the sequence useful for both USER cloning.

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]