Difference between revisions of "Part:BBa K4367010"

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Tobacco Etch Virus Protease (TEVp)

Tobacco Etch Virus Protease (TEVp) is a specific endopeptidase that recognizes the amino acid sequence ENLYFQ/G and cleaves between amino acids Q and G [1]. TEVp has a 6xHis-tag at the N-terminal, allowing for protein purification. It is also adapted for Modular Cloning as it has flanking Part-3-overhangs.

Sequence and Features

Usage

TEVp will be used as a positive control for the tests based upon using a split TEVp, and it will be used to test if the Inhibited Beta-galactosidase is functional (https://parts.igem.org/Part:BBa_K4367009). TEVp will It is possible to split TEVp into two fragments, nTEV (TEVp 1-118) and cTEV (TEVp 119-242), that are enzymatically inactive by themselves, but can complement each other to regain their enzymatic function together [2]. Split TEVp will be used in the ABI/PYL1 system and the dCas9-TEV system.

Future design considerations

There exists alternative proteases (PPVp, SbMVp, SuMMVp and HRV 3C) to TEVp that have different recognition sites and can be split, and the halves recombined to regain catalytic function [2, 3]. The proteases are displayed in Table 1.

Table 1. List of proteases (TEVp, PPVp, SbMVp, SuMMVp, HRV3C) and their recognition sites.

TEVp, PPVp, SbMVp and SuMMVp are all quite similar, as shown when their amino acid sequences are BLAST:ed against each other. HRV 3C has a very different amino acid sequence when compared to the other proteases. TEVp suffers from being somewhat insoluble in water [4], and as such HRV 3C may be a preferable option as it may be more soluble than TEV and the other proteases. Also, if it is shown that e.g dCas9-nTEV or dCas9-cTEV would misfold, or there exist other complications, these proteases could replace TEVp, provided the corresponding recognition site is also replaced in the targeted protein.

Characterization

The experimental setup tested the mixtures of X-gal and cell-lysates from S. cerevisiae transformed with TEVp or iGal. Each column corresponds to one transformation of S. cerevisiae with TEVp and each row makes up one transformation with the iGal. As negative control, no TEVp and iGal were added to the last column and row respectivly.

The first experiment was performed according to the specified experimental setup and it used MilliQ containing X-gal as buffer. The result is shown in figure 5. Going from left to right, the pictures show how the experiment looks after 1, 2, 3, and 6 days since its start.

Figure 5. An experiment mixing lysed cells that were with iGal and TEVp, with X-gal and MilliQ as buffer.

The second experiment used the same experimental setup, but the MilliQ buffer was replaced with PBS. The result is shown in figure 6. Going from left to right, the pictures show how the experiment looks after 1, 2, and 5 days since its start.

Figure 6. An experiment mixing lysed cells that were with iGal and TEVp, with X-gal and PBS as buffer.

A detailed analysis of the experiment can be found in Results, but a conclusion was that the lysed cells of S. cerevisiae contained something that degraded X-gal. This made it not possible to conclude if TEVp and/or iGal were expressed or were functional.

References

[1] David S. Waugh. TEV Protease FAQ. Available at: https://structbio.vanderbilt.edu/wetlab/private/vectors/Tev/tevnotes.Waugh.pdf

[2] Tina Fink, Jan Lonzarić, Arne Praznik, Tjaša Plaper, Estera Merljak, Katja Leben, Nina Jerala, Tina Lebar, Žiga Strmšek, Fabio Lapenta, Mojca Benčina & Roman Jerala (2019). Design of fast proteolysis-based signaling and logic circuits in mammalian cells. Available at: https://www.nature.com/articles/s41589-018-0181-6 iGEM16_Slovenia has been a source inspiration in the design of the Cell Free system and they were the ones that came up with the proof-of-principle behind the paper ‘Design of fast proteolysis-based signaling and logic circuits in mammalian cells’.

[3] Shengchen Wang, Faying Zhang, Meng Mei, Ting Wang, Yueli Yun, Shihui Yang, Guimin Zhang & Li Yi (2021). A split protease-E. coli ClpXP system quantifies protein–protein interactions in Escherichia coli cells. Available at: https://www.nature.com/articles/s42003-021-02374-w

[4] Susanne van den Berg, Per-Åke Löfdahl, Torleif Härd, Helena Berglund (2021). Improved solubility of TEV protease by directed evolution. Available at: https://www.sciencedirect.com/science/article/pii/S0168165605004840