Difference between revisions of "Part:BBa K3617001"

Revision as of 18:32, 24 October 2020

sgp130(D1-D3)-Cub

This biobrick is a part of a two-protein system that is designed for detection of human IL-6 and transduction of the signal by means of a reconstituted ubiquitin. Development of split-ubiquitin as a tool for study of protein-protein interactions in vivo was first published in 1994 and has been an essential feature in biologists’ toolbox ever since (source: https://www.pnas.org/content/pnas/91/22/10340.full.pdf). A specific mutation in the N-terminal part protects it from binding spontaneously to the C-terminal part, however, reassociation can be facilitated by binding of a pair of proteins to which ubiquitin parts are fused. Human signal transducer gp130 was expressed in yeast for the first time in 1997 and further improvements paved the way to our own chimeric transmembrane proteins (source: https://pubmed.ncbi.nlm.nih.gov/9271090/).

Sequence and Features

This biobrick consists of multiple parts; An endoplasmatic reticulum import signal peptide from the Saccharomyces cerevisiae cell wall integrity and stress response component 1 (Wsc1) receptor in S. cerevisiae, the first and third domain of human soluble interleukin-6 co-receptor soluble glycoprotein 130 (sgp130), the transmembrane receptor of Wsc1 and the C-terminal part of the split version of ubiquitin, constituting aa. 363-405. Between the three domains of sgp130 and the transmembrane domain, a GGGGS-linker was added. Between the C-terminal split ubiquitin domain, two basic amino acids (KR), and the GGGGS-linker was added.

A part of the protein constitutes of the C-terminal part (aa. 35-76) of the split ubiquitin molecule, which is fused to the reporter protein cassette LexA-VP16. Ubiquitin can be split into two parts, being the N-terminal and C-terminal ubiquitin that are able to reassociate in vivo to form active ubiquitin. This complementation was utilized so that upon interleukin-6 forming a heterotrimer with the gp130 domains of this biobrick and the IL-6 receptor domains of BBa_K3617000, the whole ubiquitin-molecule is formed, leading to activation of an intracellular deubiquitinase, thereby leading to release of Cub-bound LexA/VP16 . LexA is a DNA binding domain from Escherichia Coli (EG10533 (EcoCyc); P0A7C2 (UniProt)) and Herpes simplex virus Type 1 VP16 gene is a transcriptional activation domain. Upon complementation of split ubiquitin, the LexA-VP16 transcription factor is released and transported into the nucleus where it triggers reporter expression by promoter binding.

structure and function

The signal peptide and transmembrane domain constitute the backbone of the modular framework of the UCopenhagen 2020 team (CIDosis). These are used for localizing interleukin-1, interleukin-6 and interleukin-10 receptors 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 PPIs at the plasma membrane of S. cerevisiae.

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 (Vollmer et al. (which year) PMID: 10406952). This biobrick is intended to work together with (Biobrick?) that possesses the outer three domains of sgp130 extracellularly. Intracellularly, (Biobrick?) also consists of the synthetic transcription factor (LexA/VP16?) linked to the C-terminal of the split ubiquitin domain. BBa_K3617001 and BBa_K3617000 localizes to the same compartment, being the plasma membrane of S. cerevisiae, but will only associate upon presence of IL-6. When IL-6 is present, the extracellular domains of the IL-6R and sgp130 will associate into a heterotrimer consisting of IL-6, IL-6R, and sgp130.

Sequence optimization

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

Confocal flourescence microscopy

In order to investigate the localization of our protein, superfolding green flourescent protein was linked C-terminally to the protein product of the biobrick and expressed in S. cerevisiae, the cells were observed with confocal flourescence microscopy for visualization.

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.

The majority of investigated cells had either multiple or a single big fluorescent aggregate. This aggregate was positioned between the nucleus and the plasma membrane and can likely be attributed to the presence of inclusion bodies. It is possible that the protein may be stuck in the golgi apparatus, which is especially evident for the cells that had only one accumulation near their nucleus, in accordance to previous findings of Vollmer et al. (year) that have shown that removing the N.terminal Ig-like domain of the IL-6 receptor leads to retention of the protein in the secretory pathway. To circumvent this localization issue, one could add back the N-terminal Ig-domain of the IL6-R.

Biosensor assays

To test the functionality of the part, it was stably transformed into chromosome X site 3 of S. cerevisiae and constitutively expressed by the pTDH3 promoter together with BBa_K3617001. The latter was under constitutive expression by the pPCCW12 promoter. Together, they were also expressed with a NanoBiT luciferase, which luminesces 100 times brighter than firefly or Renilla luciferase(citation). The luciferase expression was conditionally controlled by binding of lexA-VP16 to the lexAop promoter.

After growing the cell cultures to an OD600=0,5, the cells were incubated at 30°C with different concentrations of commercial heterologously expressed IL-6 for 1, 3, 14 and 22 hours. A luminescence assay was performed to analyze the expression of luciferase after application of an industrial extraction reagent called YeastBuster to the samples, which allows for fast extraction of native proteins from yeast without mechanical disruption and enzymatic lysis, mixed with NanoBiT substrate.

Figure 4: Il-6 split ubiquitin biosensor luciferase assay.

No correlation between IL-6 concentration and luminescence intensity was observed at any incubation time. This indicates, that the biosensor does not work as intended for the concentrations and conditions of the experiment.

A similar assay was performed with the IL-10 biosensor strain also developed by the UCopenhagen 2020 team (link to biobricks).

Figure 4: Il-10 split ubiquitin biosensor luciferase assay.

Compared with the IL-10 biosensor, the amount of luminescence was between 3-10 times higher at all concentrations and incubation times for the IL-6 biosensor. This suggests that the two extracellular domains have an affinity to each other even without the presence of IL-6. This further implies that the proteins produced from BBa_K3617000 and BBa_K3617001 are localized at the same subcellular compartments. The protein product of BBa_K3617001 may also be partially degraded, after which the synthetic transcription factor, LexA-VP16, is released and re-localizes to the nucleus. The degradation may take place without any interaction with the protein product of BBa_K361700. One could verify this by integrating only the BBa_K3617001 and reporter gene into S. cerevisiae and performing an additional luciferase assay. Alternatively, one may perform a western blot with primary antibody against GFP on the strain used for the localization assays.

References

[1] Paste reference APA-style here

[2] Paste reference APA-style here

[3] Paste reference APA-style here

[4] Paste reference APA-style here

[5] Paste reference APA-style here