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  • Simplified Expression and Purification Protocol (E6901)

    The following protocol is provided as a general guideline (see Figure 5 on main product page).

    Protocol

    1. Transformation: Transform the plasmid bearing the target gene into competent T7 Express or competent cells prepared from ER2566.
    2. Cell Culture: Inoculate a freshly grown colony in LB medium containing 100 g/ml ampicillin and grow the cells at 37°C. When the OD600 reaches 0.5, induce protein expression by adding IPTG to a final concentration of 0.4 mM, and incubate at 30-37°C.
    3. Column Preparation: Equilibrate a chitin column (20 ml slurry for 1 liter culture) with 10 column volumes of Column Buffer [20 mM Tris-HCI (pH 8.5), 500 mM NaCl].
    4. Cell Harvest: Centrifuge cell culture at 5,000 x g for 15 minutes at 4°C. Discard supernatant. Resuspend cell pellet in column buffer.

    5. Loading: Break cells by sonication in Column Buffer, and centrifuge at 15,000 g for 30 minutes at 4°C. Slowly load the clarified lysate onto the chitin column (0.5-1.0 ml/minute).
    6. Washing: Wash the column with at least 20 bed volumes of Column Buffer to thoroughly remove the unbound proteins (up to 2.0 ml/minute).
    7. Adding Thiols: Quickly wash the column with 3 column volumes of Cleavage Buffer [Column buffer containing 50 mM DTT (for purification) or 50 mM MESNA (for IPL)].
    8. On-column Cleavage: Stop the flow and incubate the column at 4°C-23°C for 16-40 hours. The temperature and duration of the cleavage reaction are dependent on the on-column cleavage efficiency which can be checked by analyzing samples of chitin resin before and after cleavage.
    9. Elution: Elute the target protein with Column Buffer by continuing the column flow.
    10. Dialysis: Dialyze the target protein in to an appropriate storage buffer; this will also remove the excess thiol reagent used in the Cleavage Buffer and the co-eluted small peptide (when using pTYB21).
    11. Cleavage: To examine cleavage efficiency remove 100 μl of chitin resin and mix with 50 μl of 3X SDS Sample Buffer. After boiling for 5 minutes, analyze the supernatant on a Coomassie stained SDS-PAGE gel to determine the cleavage efficiency.
    12. Regeneration of Chitin Resin: Wash the column with 3 bed volumes of the 0.3 M NaOH (Stripping Solution). Allow the resin to soak for 30 minutes and wash the resin with an additional 7 bed volumes of 0.3 M NaOH. Wash with 20 bed volumes of water, followed by 5 bed volumes of column buffer.