Part:BBa_K2450101

TEV protease tagged with mCherry

A non-self-cleaving TEV protease sequence with an N terminal mCherry tag and a C terminal His tag. The fluorophore tag allows for relative quantification of expression. The His tag allows for purification using a nickel column.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1444
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

This part does not require any other parts to be functional.

For more information on how this part was used in our project, please see our <url="2017.igem.org/Team:Oxford">wiki</url>.

TEV protease

TEV protease crystal structure

The Tobacco Etch Virus protease is a well-characterised specific protease with a cleavage sequence of Glu-Asn-Leu-Tyr-Phe-Gln-(CUT)-Gly.

This is a non-self-cleaving protease as we wanted a constant level of TEV protease produced.

We know that TEV protease can be purified using a 6-His tag.

In our project, we have used TEV protease as both a substitute for cruzipain, another specific protease, and as a signal amplifier after simulated cruzipain cleavage. This is because it is a very common endopeptidase used in biotechnology, so we could be sure of its activity.

mCherry

mCherry is a red fluorophore derived from Discosoma. It is a very photostable monomer, and is commonly used to follow translation of proteins. It has a peak excitation/emission at 587 nm/610 nm, making it compatible with other colour fluorophores such as CFP and YFP. It matures very quickly, and fluoresces in a matter of minutes after translation.

It can also participate in FRET experiments.

Linker

We used a linker from the Wu et al. paper with the amino acid sequence GSKGP.

References

PDB: 1Q31

Francesca Cesaratto, Oscar R. Burrone, Gianluca Petris, Tobacco Etch Virus protease: A shortcut across biotechnologies, In Journal of Biotechnology, Volume 231, 2016, Pages 239-249

Tropea J.E., Cherry S., Waugh D.S. (2009) Expression and Purification of Soluble His6-Tagged TEV Protease. In: Doyle S.A. (eds) High Throughput Protein Expression and Purification. Methods in Molecular Biology, vol 498. Humana Press

Xudong Wu, Di Wu, Zhisheng Lu, Wentao Chen, Xiaojian Hu, and Yu Ding, “A Novel Method for High-Level Production of TEV Protease by Superfolder GFP Tag,” Journal of Biomedicine and Biotechnology, vol. 2009, Article ID 591923, 8 pages, 2009. doi:10.1155/2009/591923