Purification of His-TEV(S219V)-Arg

BL21-RIL cells containing pRK793 are grown at 37 degrees in L-broth containing 
100 ug/ml ampicillin and 30 ug/ml chloramphenicol. When the cells reach mid log 
phase (OD600 ~ 0.5), IPTG is added to a final concentration of 1 mM and the temp-
erature is reduced to 30 degrees.  After 4 hrs of induction, the cells are colle-
cted by centrifugation.

Cells containing His-TEV(S219V)-Arg were resuspended in 10 ml of 50 mM PO4 
(pH 8.0) + 100 mM NaCl + 10% glycerol + 25 mM imidazole (lysis buffer) per 1 gram
of wet weight cells.

Cells were then lysed using a Gaulin cell homogenizer @ 10,000-10,500 psi for 3 
passes.

The lysate volume was then determined and 5% polyetheleneimine (adjusted to pH 
7.9 with HCl) was added to a final concentration of 0.1%.

The lysate was then mixed by inversion and centrifuged at 15,000 x g for 30
minutes.

The supernatant was then applied onto a Ni-NTA column equilibrated with lysis 
buffer (I suggest 1.0 ml of resin per 0.5 g of wet weight cells).

The column was washed with 7 column volumes of lysis buffer, and then the TEV was
eluted using a 10 column volume gradient to 50 mM PO4 pH 8.0 + 100 mM NaCl + 10% 
glycerol + 200 mM Imidizole.

Appropriate fractions were examined on SDS-page and pooled.
EDTA and DTT were then added to the solution to 1mM.

The sample was then concentrated in a stirred cell using a YM10 membrane.

The TEV was then loaded onto a S-100 column in 25 mM PO4 pH 8.0 + 200 mM NaCl + 
10% glycerol + 2mM EDTA + 10 mM DTT using 3% of the column volume.

Samples were then examined on SDS-page and pooled appropriately.

TEV was concentrated to approximately 1.0 mg/ml, glycerol was added to a final 
concentration of 10% (v/v), and then the aliquots were flash frozen with liquid 
nitrogen and stored at -80C

Yield is approximately 10 mg of protein per gram of wet cell paste (ca. 30 mg/liter)

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