Part:BBa_K3147006

mutant TEV (PE10) protease with TEV-cleavable maltose binding protein

I. Part BBa_K3147006 function

The 2019 Montpellier iGEM team made this construction in order to be able to compare the basal activity of the TEV protease Part:BBa_ K1319004 with the mutant TEV protease TEVPE10 [3]. A modified TEV cut-off site was added between the MBP and the protease. MBP increases the solubility of the fusion protein [1], preventing the aggregation of the protein of interest. This stabilizes the expression and the sequence of the produced MBP does not have a signal peptide, which allows the protein to remain in the cytosol. The TEV cutting site allows self-cleavage of MBP from TEV once the protein is produced [2].

Figure 1: Construct Design: MBP-TEVcs-TEVPE10 with modified TEV cutting site.

II. Proof of function

The construction was cloned by Gibson Assembly in a pOUT18 backbone under the control of a Tet-on promoter in order to control its expression. The TEV cutting site used is the mutated site: ENLYFE/G this mutation allows theoretically the mutant TEV to better cleave this sequence according to this source [3].

Figure 2 : MBP-TEVPE10 reporter gene in its backbone pOUT18.

The experimental approach that has been used to test the protease activity is to compare the fluorescence restoration rate of sfGFP compared to MBP-TEV and MBP-TEVPE10. In this experiment, basal controls of maximum and minimum fluorescence of the reporter genes were used. Fluorescence data are obtained from plate reader at 37°C with E.coli NEB10B. The protease is expressed by inducing the Tet promoter with 50 ng/mL of aTc (anhydrotetracycline).

Figure 2: Comparison of the activity of TEVPE10 to the wild-type TEV by measuring the fluorescence of a reporter fused to a cleavable proteolysis tag

We can see that the TEVPE10 is more efficient than the wild-type TEV. We believe this is because our wild-type TEV is not the same one in the article where we found this mutant.

Reference

[1]Raran-Kurussi, Sreejith, et David S. Waugh. 2012. « The Ability to Enhance the Solubility of Its Fusion Partners Is an Intrinsic Property of Maltose-Binding Protein but Their Folding Is Either Spontaneous or Chaperone-Mediated » éd. Bostjan Kobe. PLoS ONE 7(11): e49589.

[2]Shih, Y.-P. 2005. « Self-Cleavage of Fusion Protein in Vivo Using TEV Protease to Yield Native Protein ». Protein Science 14(4): 936‑41.

[3] Yi, L. et al. 2013. « Engineering of TEV Protease Variants by Yeast ER Sequestration Screening (YESS) of Combinatorial Libraries ». Proceedings of the National Academy of Sciences 110(18): 7229‑34.

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 BsaI site found at 77
  Illegal SapI.rc site found at 1868