Editing TEV Purification (section)

== <br>Expression &amp; Purification of His-TEV(S219V)-Arg  ==

<br>1. Grow BL21-RIL cells containing pRK793&nbsp; at 37 °C in L-broth containing 100 ug/ml ampicillin and 30 ug/ml chloramphenicol.<br>2. 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.<br>3. After 4 hrs of induction, the cells are collected by centrifugation.<br>4. Dissolve the cell pellet 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 cell paste.<br>5. Lyse the cells. <br>6. Add 5% polyetheleneimine (adjusted to pH 7.9 with HCl) to a final concentration of 0.1%.<br>7. Mix by inversion and then immediately centrifuge at 15,000 x g for 30 minutes.<br>8. Apply the supernatant to a Ni-NTA column equilibrated with lysis buffer, using ca. 2 ml of resin per gram of wet cell paste.<br>9. Wash the column with 7 volumes of lysis buffer, and then elute the TEV protease with a linear gradient of lysis buffer to 50 mM PO4 (pH 8.0) + 100 mM NaCl + 10% glycerol + 200 mM Imidizole in 10 column volumes.<br>10. Pool the appropriate fractions and then add EDTA and DTT to a final concentration of 1 mM each.<br>11. Concentrate the sample in a stirred cell using a YM10 membrane.<br>12. Load the sample onto an'''S-100''' column equilibrated with 25 mM PO4 (pH 8.0) + 200 mM NaCl + 10% glycerol + 2mM EDTA + 10 mM DTT (sample volume = 3% of the column volume).<br>13. Pool the appropriate fractions.<br>14. Concentrate the protease to ca. 1 mg/ml and flash freeze with liquid nitrogen.<br>Store at –80 °C.<br>The final yield should be approximately 10 mg of protein per gram of wet cell paste (ca. 30 mg/liter)<br> 

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