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:

This part is ment to function as a IL-6 receptor together with BBaK3617001. Compared to the human receptor the extracellular domains are severely truncated and the intracellular domains are replaced with our alternative signal transduction designs.

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 of the receptor (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.

Sequence optimization

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]

Confocal flourescence microscopy

Superfolding green flourescent protein was linked C-terminally to the protein and the cells where observed with confocal flourescence microscopy.

figure 3a: Pictures were taken with a 150 μm pinhole. The image shows both a faint localisation in endoplasmatic reticulum, and at the membrane, but most of the protein ends up in inclusion bodies/vacoules

figure 3b: Pictures were taken with a 150 μm pinhole. Here the inclusion bodies are also evident in the brightfield image.

figure 3c: Pictures were taken with a 150 μm pinhole. Flourescence in inclusion bodies and very faintly at membrane and around nucleus

Most of the cells had either multiple or a single big flourescing aggregation positioned in the middle between the nucleus and the plasma membrane. This is most probably inclusion bodies. Especially for the cells that only had a single accumulation near the nucleus, we speculate that the protein might be stuck in the golgi apperatus. This would fit well with the findings of Vollmer et al. (DOI: 10.1046/j.1432-1327.1999.00511.x), that removing the N-terminal Ig-like domain of the IL-6R leads to retention in the secretory pathway and possible misfolding when expressing the IL-6R in P. pastoris. Next step in order to improve localization would be to put back the N-terminal Ig-domain.

Biosensor assays

To test the functionality of this part (BBa_K3617000), it was stably transformed into chromosome x site 3 of S. cerevisiae and constitutively expressed by the pTDH3 promoter together with BBa_K3617001 under constitutive expression by pPCCW12 promoter and with nanoluciferase (citation??) under control by lexAop promoter. The cells were incubated at 30°C at a OD600=0,5 with different concentrations of commercial heterologously expressed IL-6 for 1, 3, 14 and 22 hours. We then did a luciferase assay by measuring luminescence after adding YeastBuster with 1% nanobit substrate to the samples.

figure 4: Luciferase assay.

We did not observe any correlation between interleukin-6 concentration and luminescence intensity at any incubation time. Instead, the amount of luminescence were in all cases very high compared to other strains that we measured - also for the mock experiment without any added interleukin-6. This might suggest that the two extracellular domains actually have an affinity for eachother even without the presence of IL-6, this would further imply that the two proteins, BBa_K3617000 and BBa_K3617001 are localizedd to the same subcellular compartments. Another possible explanation is that BBa_K3617001 is partially degraded after which the synthetic transcription factor is released and relocates to the nucleus. The degradation of BBa_K3617001 might even happen without any interaction with BBa_K361700. This could then be further investigated by integrating only the BBa_K3617001 and the reporter into the yeast and then doing another luciferase assay otherwise one could make a westernblot with anti-GFP on the strain used for the localization assays.