Difference between revisions of "Part:BBa K3139012"

Latest revision as of 07:55, 2 October 2024

TEVpS

Usage and Biology

TEV is a member of the Poty viridae family responsible for infections of many different plants species of Solanaceae including N.tabacum ,One of the most important TEV proteases is Nuclear Inclusion protein a(Nla).TEVpS is part of the Nuclear Inclusion a(Nla) enzyme.It is an efficient and specific protease, able to cleave its substrate ENLYFQS between the Q and S residues, leaving an ENLYFQ-tail on the C-terminus of the protein encoded upstream and a serine residue on the N-terminus of the downstream-encoded protein. Nowadays TEVpS is a unique endopepidase largely exploited in biotechnology from industrial applications to in vitro and in vivo cellular studies. In our project, we use it to secrete cutting enzyme, which can specifically recognize cleavage site on our designed fusion protein sequence and cut it into several individual anti-plasmodium peptide.

Characterize

We transferred the plasmid into BL21 strain and cultured it in LB medium to obtain supernatant after ultrasonication. Then we purified our target His-tagged protein by using Ni-NTA magnetic beads. It was predicted that TEVp cleavage would not be so complete that nine different sizes of protein bands and TEVp's own bands would appear. In the preliminary experiment, we electrophoresised the purified protein on sodium dodecyl sulfate (SDS) -12.5% (wt/vol) polyacrylamide gel, followed by Western Blot to verify the expression and cleavage effect of this plasmid in the bacteria. In the result, the band of fusion protein (75,1kDa) in sample without TEVp is deeper than the one in sample with TEVp, which strongly proves that our plasmid could express and cleave to produce proteins(Fig.1).
Fig.1. Preliminary verification of the effect of cleavage.
Lane 1: protein which purified from the sample of bacteria expressing effector and TEVp simultaneously.
Lane 2: protein which purified from the sample of bacteria expressing Effector only.

Comparing the results of fresh bacteria liquid and the results of bacteria liquid stored at 4 degrees for 4 days, we surprisingly find that : Both samples have the obvious band of TEVp （29.5kDa）.However, the band of large protein existed only in the fresh sample, not in the sample stored for 4 day while the smallest monomer protein (7.2kDa) produced by complete cleavage exited only in the sample stored for 4 day, which suggests that the second time cut was more complete than the first one (Fig.2). This result proves that TEVp was still able to perform the cutting function efficiently even at 4 °C.
Fig.2 Verification of the efficiency of cleavage.
Left: protein purified from sample of fresh engineered bacteria.
Right: protein purified from sample of engineered bacteria which have been stored in 4℃ for 4 days.

Fig.3 Mass spectrometry analysis of TEVp

The mass spectrometry analysis (Figure.3) shows that the TEVp was detected.

Improvement by AFCM-Egypt 2024

TEV protease, a crucial enzyme in molecular biology, has been widely used for protein purification and analysis. Also, it has high specificity and efficiency in recognizing and cleaving peptide bonds between specific amino acids sequence known as TEV cleavage site (TCS).

We tried to improve this part (BBa_K3139012) which was designed by iGEM19_NAU-CHINA. However, controlling TEV activity has been a longstanding challenge.To overcome this limitation, we've Integrated a CRISPR-dCas9 technology into a novel synthetic receptor based-system which ensures that TEV protease is activated only following the dimerization of the two receptor chain specific conditions. The dimerization of the receptor is conditioned upon the presence of VEGF, a substance elevated in wound injuries, particularly burns. This conditional activation mechanism significantly enhances the safety and precision of TEV protease-mediated protein release.

For more strict control over TEV protease, we've divided TEV enzyme into two non-functional domains: C-terminal domain (C-TEV) and N-terminal domain (N-TEV). Each domain is linked to a different chain of the synthetic receptor. The assembly of the two domains of TEV to form a catalytically active version of TEV protease is conditioned by the binding of VEGF to the receptor that mediates the dimerization of the two chains of the receptor.

By applying this design we could ensure the specificity of TEV protease, preventing unintended receptor activity to reduce VEGF-independent basal activity

This figure illustrates VEGF dependent dimerization of the two domains of TEV and its proteolytic activity for releasing of dCas9 .

Characterization by AFCM-Egypt2024

Dry lab Characterization by AFCM-Egypt 2024

To illustrate TEV protease assembly, we used alpha fold 3 to show the interactions between its C and N domains.

Alignment Plot

3D structure of TEV protease

The alignment plot reflects that the receptor’s 3D structure has positive alignment with the experimental structures used in alpha fold 3. The results indicate favorable protein structure. Moreover, we calculated the binding stability through measuring the difference in Gibbs free energy (ΔG) and the predicted dissociation constant (M) at normal body temperature between TEV domains by the prodigy haddock tool. The ΔG was -23.7 kcal mol-1, and M=1.8e-17 which indicates a very stable and high affinity between both domains .

Characterization by Mathematical Modeling by AFCM-Egypt 2024

The model provides the activation kinetics of the TEV protease which occurs subsequent to the binding of VEGF to our receptor allowing the dimerization process for our receptor chains to take place. The result shows sufficient TEV protease activation based on parametric values from literature [1].

Graph (1). Illustrates the dimerization level (Blue line) that reaches steady state upon binding of VEGF to its receptor to activate TEV protease (Red line), The activation level of TEV protease reaches (14) to release d-Cas9 system .

Reference

[1]Cesaratto F , Burrone O R , Petris G . Tobacco Etch Virus protease: A shortcut across biotechnologies[J]. Journal of Biotechnology, 2016, 231.
[2]Jianguo Y , Xiaqing X , Nan X , et al. Polyprotein strategy for stoichiometric assembly of nitrogen fixation components for synthetic biology[J]. Proceedings of the National Academy of Sciences, 2018:201804992-.

Sequence and Features