Part:BBa_K2969000

TEV protease

TEV protease is a highly specific cysteine protease from Tobacco Etch Virus that recognizes the amino-acid sequence Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser) and cleaves between the Gln and Gly/Ser residues. It is often used for the removal of affinity purification tags such as maltose-binding protein (MBP) or poly-histidine from fusion proteins. In our project we insert the recognition sequence into the transcription factors so that the expression of protease can inhibit the action of transcription factors.

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
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 319
  Illegal SapI.rc site found at 667
  

Characterization

We inserted the TEV protease recognition sequence between N-terminal and C-terminal of CI434 so that it can be cleaved by TEV and then lose activity. We put the reporter gene sf-GFP under the promoter of CI434 so that when TEV protease cleaves CI434, the expression of sf-GFP will rise. We use pTac to regulate the expression of TEV protease. Induced by different concentrations of IPTG, we get the cleavage effciency of TEV protease through the fluorecence of sf-GFP.

Figure 2:The cleavage efficiency of TEV to CI434-TEVsite induced by a series of concentrations of IPTG

Reference

Voigt, C. A. and Fernandez-Rodriguez, J. , Post-translational control of genetic circuits using Potyvirus proteases Jesus, 2016, 44(13): 6493-6502