Part:BBa_K3617001

sgp130(D1-D3)-Cub

Long description

This part was used to localize the sgp130(D1-D3)-C-ubiquitin receptor module of the human IL-6 biosensor in yeast.

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 referred to as Wsc1 while the gene is called SLG1 what do we call it?) receptor in S. Cerevisiae.
• The first and third domain (D1-D3; aa 22-323) of human IL-6 co-receptor soluble glycoprotein 130 (sgp130).
• The transmembrane domain of Wsc1 (aa 327-351)
• C-terminal part of a split version of ubiquitin. (aa 363-405)
• Between the three domains of sgp130 and the transmembrane domain we added a GGGGS-linker and between the transmembrane domain (Wsc1) and the C-terminal split ubiquitin domain we added two basic amino acids; KR, and the GGGGS-linker again. [Jon: do you think we should explain the rational of these gene engineering choices or is that redundant?]

The C-terminal part (Cub) is the amino acids 35-76 of the split ubiquitin molecule and has the reporter protein cassette LexA-VP16 fused to Cub. LexA is a DNA binding domain from E. Coli (EG10533 (EcoCyc); P0A7C2 (UniProt)) and Herpes simplex virus Type 1 VP16 gene is a transcriptional activation domain.

The LexA-VP16 protein cassette is used in yeast two-hybrid method (Y2H) to assay protein-protein interaction (PPI), where the LexA-VP16 is dissociated from the Cub by deubiquitinase when PPI occurs. The LexA-VP16 transcription factor will then be transported into the nucleus where it will trigger the expression of reporter genes.

The sequence was codon optimized for S. cerevisiae using Benchling.

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 mediating the binding of the receptor to interleukin-6 as seen in crystal structures (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_K3617001 (this BioBrick) and BBa_K3617000 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. Unfortunately, our assays indicated that the BioBricks do not work together as intended.

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 1515
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]