===== Cabrita TEV Purification protocol =====


Two point mutations were introduced to produce the double mutant L56V/S135G. The mutations were introduced using the QuikChange Site Directed Mutagenesis kit (Stratagene).(([[http://onlinelibrary.wiley.com/doi/10.1110/ps.072822507/abstract;jsessionid=4269BED8C9677045F562FB78F91A872F.f04t02|Cabrita et al. (2007) Protein Science 16, 2360-2367]]))
 

  * The proteins were expressed in BL21pRIPL //E. coli// (Stratagene).
  * 1 L of culture.
  * isopropyl β-D-thiogalactopyranoside (IPTG) (0.5 mM) induction. 
  * The cells were then lysed using sonication. 
  * The sample was then applied to 1 mL of Ni-NTA agarose (GE Healthcare) pre-equilibrated with Buffer A((25 mM sodium phosphate (pH 8.0), 10% (v/v) glycerol, 200 mM NaCl, and 25 mM imidazole)). 
  * The protein was eluted with Buffer B((25 mM sodium phosphate (pH 8.0), 10% (v/v) glycerol, 200 mM NaCl, and 500 mM imidazole)). 
  * The eluted protein was then applied to a Superdex 75 (16/10) column in Buffer C((25 mM sodium phosphate (pH 8.0), 10% (v/v) glycerol, 200 mM NaCl, and 5 mM β-mercaptoethanol)). 
  * The eluted fractions containing TEV were pooled, flash-frozen in liquid nitrogen, and stored at −80°C.