Part:BBa_K3617001:Design

sgp130(D1-D3)-Cub

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]

Design Notes

Considerations for design:

The biobrick only includes three out of six extracellular domains of the sgp130 co-receptor. The three domains that we are not including (D4 to D6) are about 44Å, 44Å and 49Å long respectively according to PDBID: 3l5H. According to the same structure the domains are a bit stacked so that the distance between the start of D4 and end of D6 is actually only about 100Å.

Xu et al. [ref_id] has determined the structure mentioned and in their report they support the view that the bend between D4 and D5 is important for bringing the two intracellular and TM domains close enough to induce signaling through JAK phosphorylation. If this is true we might have problems with substituting the three membrane proximal domains. The three proximal domains evidently play a vital role in transduction but they aren’t that important in binding, except by providing conformational flexibility (Kurth et al.). 

gp130 to membrane linker:

The distance between the C-terminals of the to D3 domains is about 71Å according to PDBID 1P9M.

1. We could connect these very closely (with a small linker eg. 8 residues) to the PM and then have long flexible linkers (about 15-20AA) on the inside so the split proteins can meet even though the TMDs will be about 70 Å apart. Pros: that we will not need to express the three proximal domains and we will know the distance as well as the orientation of the two TMDs. Cons: less flexibility of extracellular domain maybe resulting in smaller binding affinity and that we now don’t have "tall" receptors.
2. We try to express all 6 domains of the ectodomain. Pros: if it works, we will have smaller distance between TMDs, and the extracellular domain will work in the same way it does naturally. Cons: it is a very big protein to express and insert in the membrane.
3. 3. We add a very long linker extracellularly to achieve the same height and flexibility as natural receptor. Pros: we are possibly elevating binding affinity and efficiency of complex formation. Cons: we will not know distance between TMDs and it can possibly be very big, also we don't know if we can express such long linkers (about 65 AA if helical, less if disordered).
4. 4. Entire new approach where we don't need association of hexameric complex but only heterotrimer: we fuse D2 and D3 of gp130 to detection device 1 and D2 and D3 of IL-6R to detection device part 2. sIL-6R∆Ig should be as biologically active as Wt in regards to receptor activation (<a href="doi:%2010.1046/j.1432-1327.1999.00511.x">DOI: 10.1046/j.1432-1327.1999.00511.x</a>) it might have a higher propensity of getting stuck in secretory pathways...

LexA-VP16:

In some papers they have a small scar from the recombination technique, but maybe this also works as a linker; one could try adding a small linker or the same scar, just to use something that has proven to work, but it is unlikely that it is something strictly important since there hasn’t been found any research advocating for adding a linker.

Source

This parts contains genomic sequences from homo sapiens (sgp130), E. coli (LexA) and human herpesvirus (VP16).