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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1444
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
This part does not require any other parts to be functional.
TEV protease
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.
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
Functional Parameters
| None |