Difference between revisions of "Part:BBa K2450101"
(→‎Usage and Biology)
Line 10: Line 10:
 
===Usage and Biology===
 
===Usage and Biology===
  
−
TEV protease is a well-characterised specific protease from the Tobacco Etch Virus, with a cleavage sequence of Glu-Asn-Leu-Tyr-Phe-Gln-(CUT)-Gly.
+
This part does not require any other parts to be functional.
 +
 
 +
===TEV protease===
 +
 
 +
[[File:Oxford_2017_TEV_protease.png|300px|thumb|left|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.
 
This is a non-self-cleaving protease as we wanted a constant level of TEV protease produced.
  
−
We have used TEV protease as both a substitute for cruzipain, another specific protease, and as a signal amplifier after simulated cruzipain cleavage.
+
We know that TEV protease can be purified using a 6-His tag.
  
−
This part does not require any other parts to be functional.
+
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.
 +
 
 +
[link:2017.igem.org/Team:Oxford]Our Wiki[/link]
 +
 
 +
===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
  
  
−
<!-- Uncomment this to enable Functional Parameter display
 
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K2450101 parameters</partinfo>
 
<partinfo>BBa_K2450101 parameters</partinfo>
−
<!-- -->
 

Revision as of 10:45, 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.

[link:2017.igem.org/Team:Oxford]Our Wiki[/link]

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