===== Expression & Purification of His-TEV(S219V)-Arg =====


S. Cherry, J. E. Tropea, D. S. Waugh(([[http://link.springer.com/protocol/10.1007%2F978-1-59745-196-3_19|Tropea et al. (2009) Methods in Molecular Biology 498, 297-307]]))

  * BL21-RIL cells containing pRK793 are grown at 37 °C in L-broth containing 100 μg/ml ampicillin and 30 μg/ml chloramphenicol.
  * When the cells reach mid log phase (OD600~0.5), IPTG is added to a final concentration of 1 mM and the temperature is reduced to 30 °C.
  * After 4 hrs of induction, the cells are collected by centrifugation.
  * Dissolve the cell pellet in 10 ml of Lysis Buffer((50 mM PO4 (pH 8.0), 100 mM NaCl, 10% glycerol and 25 mM imidazole)) per 1 gram of wet cell paste.
  * Lyse the cells. We use a Gaulin cell homogenizer @ 10,000-10,500 psi for 3 passes.
  * Add 5% polyetheleneimine (adjusted to pH 7.9 with HCl) to a final concentration of 0.1%.
  * Mix by inversion and then immediately centrifuge at 15,000 x g for 30 minutes.
  * Apply the supernatant to a Ni-NTA column equilibrated with Lysis Buffer, using ∼ 2 ml of resin per gram of wet cell paste.
  * Wash the column with 7 volumes of Lysis Buffer, and then elute the TEV protease with a linear gradient of Lysis Buffer to Elution Buffer((50 mM PO4 (pH 8.0), 100 mM NaCl, 10% glycerol and 200 mM Imidizole)) in 10 column volumes.
  * Pool the appropriate fractions and then add EDTA and DTT to a final concentration of 1 mM each.
  * Concentrate the sample in a stirred cell using a YM10 membrane.
  * Load the sample onto an S-100 column equilibrated with GF Buffer ((25 mM PO4 (pH 8.0), 200 mM NaCl, 10% glycerol, 2mM EDTA and 10 mM DTT)) (sample volume = 3% of the column volume).
  * Pool the appropriate fractions.
  * Concentrate the protease to ∼ 1 mg/ml and flash freeze with liquid nitrogen.
  * Store at –80 °C.


The final yield should be approximately 10 mg of protein per gram of wet cell paste (∼ 30 mg/liter)