Coding

Part:BBa_K1965009

Designed by: Nik Franko   Group: iGEM16_Slovenia   (2016-10-17)

Introduction

TEV protease is a highly specific 242 amino acids long, 27 kDa cysteine protease, that originates from the tobacco etch virus (TEV) of the Potyvirus genus. It has a target recognition sequence of seven amino acids, ENLYFQ-S/G, where cleavage occurs after the glutamine residue and is denoted by the – symbol, and the final residue of the recognition sequence can be either S or G, denoted by the / symbol. This substrate sequence is scarcely represented in the proteome. TEV protease is therefore relatively non-toxic[1] and can be safely expressed in host cells. Due to this non-toxicity and its high cleavage specificity, TEVp is an attractive protease for use in several biotechnological applications, such as the removal of the affinity tags from recombinant proteins.

Despite its widespread use in the biotechnology, TEVp also displays some shortcomings, the most prominent of them being self-cleavage. Substitution of Ser-219 with Val or Pro [2] or a replacement of the C-terminal sequence MSELVYSQ with the sequence MNEGGGLE [2] decreased the self-cleavage of TEVp and thereby increased its activity.

Characterization

To test these two proteases we used a cleavable firefly luciferase (fLuc) reporter with an appropriate cleavage sequence inserted in a permissible site. We observed a significant decrease in the fLuc activity upon coexpression of the reporters with their corresponding proteases, whereas the coexpression of reporters with an orthogonal protease resulted in a much lower decrease of fLuc activity ( 1 ).

Activity and orthogonality of TEVp variants.
HEK293T cells were transfected with the indicated fLuc:TEVs and TEVp variant constructs. Luciferase activity was measured 24h after transfection. The results are presented as normalized firefly luciferase activity (RLU).

Therefore we performed a viability test for expression of all three TEVp variants in HEK293T cells. Even after transfection with a high amount of the plasmid for each respective protease, the cells showed high viability, with practically no difference when compared to control transfections ( 2 ).

Toxicity test of different TEVp variants for HEK203 cells.
Cells were transfected with plasmid DNA for different TEVp variants and counted two days later. Prior to counting cells were stained with trypan blue to determine the percentage of dead cells.

References

[1]Parks, T. D., Leuther, K. K., Howard, E. D., Johnston, S. A., & Dougherty, W. G. (1994). Release of proteins and peptides from fusion proteins using a recombinant plant virus proteinase. Analytical Biochemistry, 216(2), 413–7. doi:10.1006/abio.1994.1060
[2]Cesaratto, F., López-Requena, A., Burrone, O. R., & Petris, G. (2015). Engineered tobacco etch virus (TEV) protease active in the secretory pathway of mammalian cells. Journal of Biotechnology, 212, 159–66. doi:10.1016/j.jbiotec.2015.08.026

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 NgoMIV site found at 103
    Illegal NgoMIV site found at 1447
    Illegal NgoMIV site found at 1468
    Illegal AgeI site found at 1171
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 1538
    Illegal SapI site found at 1694
    Illegal SapI.rc site found at 1353


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