Protein Expression and Purification of MBP-T antigen (131-259)

Comments: T antigen (131-259) is an MBP-Hisx6-fusion protein constructed by Laura Mizoue (refer to the wisdom page for details on the construct).
  1. Pick a single colony from freshly transformed cells (BL21 (DE3) is the expression host) into 2 ml of LB medium containing Kanamycin (20 µg/ml). (I usually pick four colonies for the safe side)
  2. Grow for 6 to 8 hours at 37 °C
  3. Inoculate 0.2 ml of culture into 25 ml of LB containing Kanamycin and grow at 37 °C, O/N. (For 6 L prep. I usually inoculate 1 ml into 150 ml medium. For 6 L labeling prep. set up 2 x 150 ml of cultures into M9 labeling medium)
  4. Transfer 25 ml culture into 1 L LB medium (or 50 ml of M9 culture into 1 L M9-labeling medium) containing Kan. Grow cells at 37 °C until the cells reach an OD600 of 0.8.
  5. Induce cells with IPTG to a final concentration of 1 mM and grow for another 3 to 4 hours at 37 °C.
  6. Harvest the bacteria by centrifugation at 7000 rpm (20 min) and freeze the cells at -20 °C for lysis/purification the following day.
  7. Thaw the cells and resuspend in Buffer A (~10 ml/1 L culture). Add lysozyme to a final concentration of 0.5 mg/ml and 2 tablets of protease inhibitors (P.I.) per 60 ml lysate.
  8. Incubate the cells ice for 10 min and add NP-40 and MgCl2 to a final concentration of 1% and 1 mM, respectively.
  9. Sonicate cells at 20 s burst with 30 s break for 10 min at power 4 (by the end of the procedure the solution should not be very viscous). After sonication save 100 ml of sample, spin down at 12 K rpm and save the sup. and pellet for SDS-PAGE
  10. Centrifuge the E. Coli extract at 22,000 rpm for 30 min
  11. To remove nucleic acids add poly-ethyleneimine to the supernatant at a final concentration of 0.2% and mix by inverting the tube and incubate at 4 °C for 10 min. Spin down at 22,000 rpm for 20 min to remove the precipitated nucleic acids.
  12. Load the supernatant on to a pre-equilibrated Ni-NTA column (the column should be equilibrated with Buffer A for at least 2 column volumes)
  13. Use a gradient program in FPLC or AKTA to purify the fusion protein. Set the gradient to be at least 2 column volumes
  14. After collecting fractions clean the column with 1 column volume of Buffer B followed by storage with 20% EtOH.
  15. Pool fractions (judged from SDS-PAGE analysis) and add thrombin (200U for 6 L prep.) and dialyze sample against buffer C O/N in cold room. Make sure the cleavage is complete. If it is not complete let the reaction go at room temperature with another addition of 100 ul of thrombin.
  16. Pass the sample over pre-equilibrated benzamidine and Ni-NTA beads (set up gravity column)
  17. Collect FT and four washes of 25 ml using Buffer A containing 30 mM Imidazole. Run SDS-PAGE and select fraction containing T-ag (~15 kDa) and set-up dialysis against buffer C O/N and proceed for MonoS column purification.
  18. Clean the monoS column with 4 column volumes of Buffer E and equilibrate with 4 column volumes of Buffer D
  19. Load the fractions using super loop and set-up a gradient program for 10 column size
  20. The protein elutes as a single sharp peak. Collect the fraction and freeze the protein at -80 °C until further use.
Buffer A

50 mM HEPES, 0.3 M NaCl, 1 mM BME, 10 mM Imidazole, 1 tablet PI/L, pH 7.5

Buffer B

50 mM HEPES, 0.3 M NaCl, 1 mM BME, 300 mM Imidazole, 1 tablet PI/L, pH 7.5

Buffer C

50 mM HEPES, 0.3 M NaCl, 1 mM BME, pH 7.5

Buffer D

50 mM HEPES, 50 mM NaCl, 1 mM DTT pH 7.2

Buffer E

50 mM HEPES, 1 M NaCl, 1 mM DTT, pH 7.2