Part:BBa_K3617002

Interleukin-10 receptor type 1 - C-ubiquitin-LexA-VP16

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

• The first 21 amino acids (Signal peptide for import to endoplasmic 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 second domain (aa 22-235) of human interleukin-10 receptor subunit α (IL-10RI).
• The transmembrane domain from Wsc1
• C-terminal part of a split version of ubiquitin. This means the last 42 amino acids (aa 35-76) of the ubiquitin protein. When binding to the N-terminal part of the split-protein, an endogenous deubiquitinase can cleave the polypeptide chain just C-terminally of the C-terminal part of the split protein.
• Between the IL-10R1 domains and the transmembrane domain we added a flexible 2XGGGGS linker (cite: PMID: 23026637) and between the transmembrane domain and the C-terminal split ubiquitin domain we added two basic amino acids; KR, and the 2XGGGGS linker again.

Expected function of the protein

This part is meant to function as a IL-10 receptor together with BBaK3617003. Upon complementation of the split ubiquitin halves, the synthetic transcription factor lexA-VP16 which the relocates to the nucleus and mediates transcription of genes controlled by promoters with Lexo sites.

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 Wsc1 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 interleukin-10 receptor subunit α domains are mediating the binding of the receptor to interleukin-10 - as seen in crystal structures of the receptor (see fig. 1). This biobrick is intended to work together with BBaK3617003which has the outer two domains of the IL-10 receptor subunit β extracellularly and the N-terminal part of split ubiquitin intracellularly. We hypothesized that BBa_K3617002 (this biobrick) and BBaK3617003would both localize to the same membrane but that they would be dissociated in the absence of interleukin-10. In the presence of interleukin-10, we imagined that the extracellular domains of the two parts; IL-10Rα and IL-10Rβ, would associate into a heterotrimer consisting of IL-10, IL-10Rα and IL-10R&beta. 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
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 628
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1269
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Confocal flourescence microscopy

Superfolding green fluorescent protein (sfGFP) was linked C-terminally to the protein and the cells were observed with confocal fluorescence microscopy.

figure 2a: Pictures were taken with a 150 μm pinhole. The image shows localization of the protein at the membrane, and a few inclusion bodies

figure 2b: Pictures were taken with a 150 μm pinhole. The image shows localization to the membrane as well as the endoplasmatic reticulum

figure 2c: Pictures were taken with a 150 μm pinhole. Flourescence here is mainly found around nucleus and in a reticulum throughout the cell which might indicate localisation to the ER

Most of the cells had fluorescence at the plasma membrane and around the nucleus in what looks to be the endoplasmatic reticulum. The localization to other subcellular compartments than the plasma membrane, ie. endoplasmatic reticulum, might be a result of a very high expression rate as the biobrick was put under the constitutive promoter pCCW12 and this might "clog" the secretory pathway.