Part:BBa_K4164006

TEVp

Tobacco Etch Virus protease (TEVp) is an efficient and specific protease from Tobacco Etch Virus (TEV) with a size of 27 kDa.Despite its high specificity, TEVp can tolerate a wide variety of residues in positionP1' (the first amino acid downstream the cleavage site) of the sub-strate, making it the best protease for endoproteolytic removal of affinity tags.

In addition, TEVp is the only protease that combines several useful features for biotechnological applications, from easy and economical production to availability of open-source vectors and mutants, making it suitable for several different in vitro and in vivo applications. TEVp belongs to the family of C4 peptidases and is structured as a two-domain antiparallel ß-barrel fold, typical of trypsin-like proteases.The TEVp specific recognition motif (TEVp cleavage Site, TS) is only 7 amino acids-long: EXXYXQ-S/G (where X can be any amino acid) and proteolysis takes place between residues Gln and Ser or Gly.

In our project, we employed tobacco etch virus protease (TEV) and protein degradation tag Ssr to integrate a logic operation to control the degradation of targeted proteins (RFP) and resolve the leakage problem. The C-terminus of targeted protein was linked with a protease cleavage site (TEVENLYFQ-protease site) and a degradation tag Ssr (AANDENYAAV) in turn by linkers. When TEV was inductively expressed, it could recognize and cleave specifically at the cleavage site, and the Ssr-tagged proteins that lost the degradation tag were no longer degraded and began to work.

Fig. 1. The Tobacco etch virus protease.
(A) TEV polyprotein processed by TEVp at the indicated sites (fifilled arrowhead).
(B) TEVp crystal structure, with key residues of the catalytic site shown in red.
(C) Schematic illustration of residues on the TEVp substrate recognition pocket (S) and on the substrate cleavage site (P).
Cesaratto, Francesca et al. “Tobacco Etch Virus protease: A shortcut across biotechnologies.” Journal of biotechnology vol. 231 (2016): 239-249. doi: 10.1016/j.jbiotec.2016.06.012 Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 461
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]