Coding

Part:BBa_K3617008

Designed by: David Nrgaard Essenbk   Group: iGEM20_UCopenhagen   (2020-10-21)


GPA1 with TEV protease cleavage sequence 1

This biobrick is a modified version of the G-protein alpha subunit, GPA1, from Saccharomyces cerevisiae. It is designed to connect our receptor system with the pheromone cascade native to S. cerevisiae. Native G protein is composed of three subunits: alpha, beta and gamma. The alpha subunit undergoes conformational changes triggered by ligand binding to a GPCR, which leads to dissociation from the beta and gamma subunits. Subsequently, the beta-gamma complex triggers downstream signaling in the pheromone mating pathway. In our biosensor a TEV protease (originating from Tobacco Etch Virus) was designed to cleave the alpha subunit and trigger signaling through the pheromone pathway. To achieve this, the alpha subunit was modified to contain a cleavage site for the TEV protease. The cleavage site was inserted between residues 63 and 71 (Figure 1).

Figure 1: Structural model of GPA1 with TEV cleavage inserted at site 1. The model was created using Swiss-Model homology modelling. The red region marks the modified ENLYFQG sequence. It is located close to the N-terminal α-helix that mediates a significant amount of the binding affinity towards the β-γ complex.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

This biobrick consists of a mutant version of the Gα -protein subunit, GPA1, from the S. cerevisiae pheromone pathway. It has an inserted Tobacco etch virus (TEV) nuclear inclusion protein protease cleavage sequence: (64-65 LH → 64-70 ENLYFQG).[1]|[2]


Structure and function

In S. cerevisiae the GPA1 protein functions downstream of the G-protein coupled receptor (GPCR), Ste2/3, in the pheromone mating pathway. To facilitate cleavage of GPA1 by a TEV protease, a TEV cleavage site have been inserted in a loop present near the N-terminus of the protein (Figure 1).

<p align="justify"> Figure 2: Cleavage of modified GPA1 for the initiation of gene expression through the yeast pheromone pathway. Through cleavage of the modified G-alpha subunit containing a TEV cleavage site, the beta-gamma subunit is released, leading to binding of Ste5 to the beta-gamma subunit. This triggers activation of the yeast pheromone pathway, thereby phosphorylating and activating the transcription factor LexA-Ste12, thereby initiating gene expression.

The modified GPA1 is designed to function together with two of our modular receptor proteins with split TEV proteases intracellularly and interleukin receptors with ligand-binding sites on the outside of the membrane. The split TEV protease is a version of the TEV protease where the protein is split into two proteins, a C- and a N-terminal part. The TEV protease only possesses proteolytic activity when the two halves are brought together. In our designs the two modular receptor proteins each have either the C- or N-terminal part of split ubiquitin. Extracellular binding of the receptors to their respective biomarkers leads to complementation of the split TEV parts which then cleaves the modified GPA1 within its inserted cleavage sequence. When GPA1 is cleaved it dissociates from the G protein beta-gamma complex and that starts signaling through the pheromone pathway.



Assays

In order to test whether our modified GPA1 could mediate signaling both though the yeast pheromone pathway and by TEV cleavage, two yeast strains were constructed (Table 1; CD14.1 and CD15). These were based on an adenosine biosensor strain, developed in Sotirios Kampranis’ lab, in which the yeast pheromone pathway has been hijacked to allow for expression of a luciferase in the presence of adenosine.

Table 1: Adenosin biosensor strains. Both biosensor strains contained a galactose inducible TEV protease, the human adenosine A2a receptor, a synthetic transcription factor, lexA-ste12, which is activated as part of the pheromone pathway, and the Nanoluc luciferase reporter under control of the LexA-ste12 transcription factor. In addition, NAME1 had native GPA1, while NAME2 had our modified GPA1.

The strains were grown to OD600=0,5 and incubated at 30°C for 17h in either glucose or galactose + raffinose media, which leads to repression or activation of TEV protease expression respectively, additionally the incubation was carried out with different concentrations of adenosine added to the cultures.

Figure 3: Luciferase assay of Adenosine receptor with BBa_K3617008 or with wt GPA1. Incubation with galactose and raffinose induces expression of the TEV protease in both biosensors.

It was observed that the concentration of luciferase correlated with the concentration of adenosine in the case of the wt GPA1 biosensor grown in glucose media. Such correlation was not observed for the biosensor with modified GPA1. This might indicate that the insertion of the TEV cut site resulted in a loss-of-function that interferes with the transduction of a signal from a GPCR to the endogenous pheromone pathway.

For both strains a significant increase in the amount of luminescence was observed when the strains were incubated in galactose + raffinose media compared to incubation in glucose media. This suggests that the media or the TEV protease may affect the luciferase concentration due to unknown circumstances, and not by the cleavage of GPA1 as intended, as the wt GPA1 should not be cleaved upon induction of TEV protease expression.

References

[1] Baeumler, T. A., Ahmed, A. A., & Fulga, T. A. (2017). Engineering Synthetic Signaling Pathways with Programmable dCas9-Based Chimeric Receptors. Cell Reports, 20(11), 2639–2653. https://doi.org/10.1016/j.celrep.2017.08.044 [2] 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

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