Difference between revisions of "Part:BBa K3139012"

Revision as of 07:46, 1 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

We partitioned TEV into two nonfunctional domains and incorporated them into our innovative synthetic receptor, dCas9(C/N)-TF Syn-VEGFR-1/2 that enables us to regulate the release of TEV, thereby mitigating unintended gene silencing effects.This conditional activation mechanism significantly enhances the safety and precision of TEV protease-mediated protein release.VEGF dependant dimerization mediate assembly of the two domains into a catalytically active enzyme which will act by cleavage of the two receptor chains mediating release of dCas9.

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