Part:BBa_K5150006

While affinity tags are powerful tools for the expression and purification of recombinant proteins, they typically must be removed before conducting structural and functional studies. Fusion technology is widely used in recombinant protein production, initially designed for detection and purification. Some fusion tags enhance yield, protect against proteolysis, improve solubility, and aid folding. However, all tags can potentially interfere with the structure and function of purified proteins, making their removal advisable. The stringent specificity of viral proteases, particularly the well-characterized nuclear inclusion protease from tobacco etch virus (TEV), makes them valuable tools for effectively removing affinity tags from recombinant proteins [1].

TEV protease is a cysteine protease that exhibits high specificity both in vitro and in vivo, with no toxicity observed in bacteria or more complex organisms. The optimal recognition site for this enzyme is the sequence Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser) [ENLYFQ(G/S)], with cleavage occurring between the Gln and Gly/Ser residues. The most commonly used sequence is ENLYFQG [2]. TEV protease is employed to cleave tags from fusion proteins, and the recognition site is present in affinity purification tags such as maltose-binding protein (MBP) or poly-histidine. TEV protease has an optimal cleavage temperature of 30°C but can also function at temperatures as low as 4°C. It is characterized by high target site specificity, ease of production, and strong activity across various buffer conditions.

[1] Raran-Kurussi, S., Cherry, S., Zhang, D., & Waugh, D. S. (2017). Removal of affinity tags with TEV protease. Heterologous Gene Expression in E. coli: Methods and Protocols, 221-230.

[2] Cesaratto, F., Burrone, O. R., & Petris, G. (2016). Tobacco Etch Virus protease: A shortcut across biotechnologies. Journal of biotechnology, 231, 239-249.

[3] Boulware, K. T., Jabaiah, A., & Daugherty, P. S. (2010). Evolutionary optimization of peptide substrates for proteases that exhibit rapid hydrolysis kinetics. Biotechnology and bioengineering, 106(3), 339-346.

[4] Kostallas, G., Löfdahl, P. Å., & Samuelson, P. (2011). Substrate profiling of tobacco etch virus protease using a novel fluorescence-assisted whole-cell assay. PLoS One, 6(1), e16136.