– Tobacco Etch Virus (TEV) protease is a highly site-specific cysteine protease commonly used to remove affinity tags from fusion proteins. It recognizes the sequence Glu-Asn-Leu-Tyr-Phe-Gln↓Gly/Ser (ENLYFQ↓G/S), with cleavage occurring between the Gln and Gly or Ser residue. The most frequently used recognition site is ENLYFQG. The optimal cleavage temperature is 30 °C, though conditions such as enzyme amount, incubation time, and temperature may require optimization for different substrates. TEV protease includes an N-terminal 6×His tag, allowing for convenient removal after digestion via Ni²⁺ affinity chromatography. The recombinant enzyme consists of 231 amino acids, has a molecular mass of approximately 28.4 kDa, and is fully biologically active following purification.