purification:tev:c

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).¹⁾

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 A²⁾.
The protein was eluted with Buffer B³⁾.
The eluted protein was then applied to a Superdex 75 (16/10) column in Buffer C⁴⁾.
The eluted fractions containing TEV were pooled, flash-frozen in liquid nitrogen, and stored at −80°C.

¹⁾

Cabrita et al. (2007) Protein Science 16, 2360-2367

²⁾

25 mM sodium phosphate (pH 8.0), 10% (v/v) glycerol, 200 mM NaCl, and 25 mM imidazole

³⁾

25 mM sodium phosphate (pH 8.0), 10% (v/v) glycerol, 200 mM NaCl, and 500 mM imidazole

⁴⁾

25 mM sodium phosphate (pH 8.0), 10% (v/v) glycerol, 200 mM NaCl, and 5 mM β-mercaptoethanol