BSI wiki

S. Cherry, J. E. Tropea, D. S. Waugh¹⁾

• 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 Buffer²⁾ 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 Buffer³⁾ 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 ⁴⁾ (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)