Difference between revisions of "Part:BBa K3617004"

 
(2 intermediate revisions by 2 users not shown)
Line 16: Line 16:
 
<partinfo>BBa_K3617004 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3617004 SequenceAndFeatures</partinfo>
  
This biobrick consists of multiple parts; An endoplasmic reticulum import signal peptide from the Saccharomyces cerevisiae cell wall integrity and stress response component 1 (Wsc1) receptor in S. cerevisiae , the second and third domain of human soluble interleukin-6 receptor subunit alpha (sIL-6R), the transmembrane receptor of Wsc1 and the N-terminal part of the split version of ubiquitin, constituting the first 34 amino acids of ubiquitin. The domain possesses the Ile13Gly mutation which inhibits the spontaneous association of the two split protein halves by reducing their affinity to each other. Between the sIL-6R domains and the transmembrane domain, a flexible 2XXGGGGS linker [3] exists. Between the transmembrane domain and the N-terminal split ubiquitin domain two basic amino acids (KR) have been added together with the 2XGGGGS linker.
+
This biobrick consists of multiple parts; An endoplasmic reticulum import signal peptide from the Saccharomyces cerevisiae cell wall integrity and stress response component 1 (Wsc1) receptor in S. cerevisiae , the second and third domain of human soluble interleukin-6 receptor subunit alpha (sIL-6R), the transmembrane receptor of Wsc1 and the N-terminal part of the split TEV protease, constituting the amino acid residues 1-118. Between the sIL-6R domains and the transmembrane domain, a flexible 2XXGGGGS linker exists. Between the transmembrane domain and the N-terminal split ubiquitin domain two basic amino acids (KR) have been added together with the 2XGGGGS linker.
  
 
==Sequence optimization==
 
==Sequence optimization==
Line 22: Line 22:
  
 
==Structure and function==
 
==Structure and function==
 +
This part is designed to function as a human IL-6 receptor together with constituent sIL-6R-cTEV. Compared to the human IL-6 receptor, only two out of three extracellular domains are included, and the intracellular domains are replaced with the N-terminal part of the split TEV protease.
 +
The signal peptide and transmembrane domain constitute the backbone of the modular framework of the UCopenhagen 2020 team (CIDosis). These are used for localizing receptor proteins for interleukin-1, interleukin-6 and interleukin-10 at the plasma membrane of <i>S. cerevisiae</i> as type I single pass transmembrane proteins. As a type I transmembrane protein, the soluble interleukin receptor domains localizes extracellularly while the N-terminal part of the split protein is intracellular. Ivanusic et al. (citation) introduced the use of the signal peptide and transmembrane domain in a split-ubiquitin system for screening for protein-protein interactions at the plasma membrane in <i>S. cerevisiae.</i>
  
 +
[[Image:T--UCopenhagen--il6-tev.png|500px|thumb|<p align="justify"> '''figure 2:  When the receptors trimerize with IL-6 extracellularly, the split TEV is complemented and the transcription factor in LexA-VP16 is released and triggers expression of the reporter gene.'''</p>]]
  
==Confocal flourescence microscopy==
+
The two fibronectin type III soluble interleukin-6 receptor subunit alpha domains mediates the binding of the receptor to interleukin-6, as demonstrated on figure 1. The outer Ig-like domain of the receptor mediates other functions of the receptor.
 
+
==Biosensor assays==
+
  
 
===References===
 
===References===
 
<p> [1] Wehr, M. C., Laage, R., Bolz, U., Fischer, T. M., Grünewald, S., Scheek, S., Bach, A., Nave, K. A., & Rossner, M. J. (2006). Monitoring regulated protein-protein interactions using split TEV. Nature Methods, 3(12), 985–993. https://doi.org/10.1038/nmeth967 </p>
 
<p> [1] Wehr, M. C., Laage, R., Bolz, U., Fischer, T. M., Grünewald, S., Scheek, S., Bach, A., Nave, K. A., & Rossner, M. J. (2006). Monitoring regulated protein-protein interactions using split TEV. Nature Methods, 3(12), 985–993. https://doi.org/10.1038/nmeth967 </p>
<p> [2]  </p>
 
<p> [3]  </p>
 
  
 
<!-- Uncomment this to enable Functional Parameter display
 
<!-- Uncomment this to enable Functional Parameter display

Latest revision as of 00:28, 28 October 2020


sIL-6R-nTEV

This biobrick is a part of a 2-protein system that is designed for detection of human interleukin-6 and transduction of the signal by means of a reconstitution of a split TEV protease. The split TEV was originally developed by Wehr, M. C. et al. In 2006[[#References[1]]] for measuring protein-protein interaction. This part resembles that of sIL-6R-Nub in which the extrecellular receptor remains the human interleukin-6 receptor, but now with a split TEV protease, an N-terminal part (amino acid residues 1-118) and a C-terminal part (amino acid residues 119-242), fused with a flexible linker on the C-terminal of the receptorbound transmembrane domain (TMD). Thus, binding of the interleukin to the receptor results in a reconstitution of the two halfes nTEV and cTEV, thus rendering it active. The activated protease will cleave a recognition site on a flexible linker bound to another TMD, which releases the synthetic transciption factor LexA-VP16.

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]

This biobrick consists of multiple parts; An endoplasmic reticulum import signal peptide from the Saccharomyces cerevisiae cell wall integrity and stress response component 1 (Wsc1) receptor in S. cerevisiae , the second and third domain of human soluble interleukin-6 receptor subunit alpha (sIL-6R), the transmembrane receptor of Wsc1 and the N-terminal part of the split TEV protease, constituting the amino acid residues 1-118. Between the sIL-6R domains and the transmembrane domain, a flexible 2XXGGGGS linker exists. Between the transmembrane domain and the N-terminal split ubiquitin domain two basic amino acids (KR) have been added together with the 2XGGGGS linker.

Sequence optimization

The sequence was codon optimize for S. cerevisiae. The recognition sequences for SpeI, XbaI, NotI, EcoRI, PstI were avoided to follow the RFC10 standard.

Structure and function

This part is designed to function as a human IL-6 receptor together with constituent sIL-6R-cTEV. Compared to the human IL-6 receptor, only two out of three extracellular domains are included, and the intracellular domains are replaced with the N-terminal part of the split TEV protease. The signal peptide and transmembrane domain constitute the backbone of the modular framework of the UCopenhagen 2020 team (CIDosis). These are used for localizing receptor proteins for interleukin-1, interleukin-6 and interleukin-10 at the plasma membrane of S. cerevisiae as type I single pass transmembrane proteins. As a type I transmembrane protein, the soluble interleukin receptor domains localizes extracellularly while the N-terminal part of the split protein is intracellular. Ivanusic et al. (citation) introduced the use of the signal peptide and transmembrane domain in a split-ubiquitin system for screening for protein-protein interactions at the plasma membrane in S. cerevisiae.

figure 2: When the receptors trimerize with IL-6 extracellularly, the split TEV is complemented and the transcription factor in LexA-VP16 is released and triggers expression of the reporter gene.

The two fibronectin type III soluble interleukin-6 receptor subunit alpha domains mediates the binding of the receptor to interleukin-6, as demonstrated on figure 1. The outer Ig-like domain of the receptor mediates other functions of the receptor.

References

[1] Wehr, M. C., Laage, R., Bolz, U., Fischer, T. M., Grünewald, S., Scheek, S., Bach, A., Nave, K. A., & Rossner, M. J. (2006). Monitoring regulated protein-protein interactions using split TEV. Nature Methods, 3(12), 985–993. https://doi.org/10.1038/nmeth967