Difference between revisions of "Part:BBa K2450101"

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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.
 
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.
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===mCherry===
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mCherry is a red fluorophore derived from <i>Discosoma</i>. 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.
  
 
===References===
 
===References===

Revision as of 11:20, 28 October 2017


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.

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.

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