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).
- 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)
- Grow for 6 to 8 hours at 37 °C
- 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)
- 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.
- Induce cells with IPTG to a final concentration of 1 mM and grow for another 3 to 4 hours at 37 °C.
- Harvest the bacteria by centrifugation at 7000 rpm (20 min) and freeze the cells at -20 °C for lysis/purification the following day.
- 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.
- Incubate the cells ice for 10 min and add NP-40 and MgCl2 to a final concentration of 1% and 1 mM, respectively.
- 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
- Centrifuge the E. Coli extract at 22,000 rpm for 30 min
- 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.
- 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)
- Use a gradient program in FPLC or AKTA to purify the fusion protein. Set the gradient to be at least 2 column volumes
- After collecting fractions clean the column with 1 column volume of Buffer B followed by storage with 20% EtOH.
- 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.
- Pass the sample over pre-equilibrated benzamidine and Ni-NTA beads (set up gravity column)
- 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.
- Clean the monoS column with 4 column volumes of Buffer E and equilibrate with 4 column volumes of Buffer D
- Load the fractions using super loop and set-up a gradient program for 10 column size
- The protein elutes as a single sharp peak. Collect the fraction and freeze the protein at -80 °C until further use.
50 mM HEPES, 0.3 M NaCl, 1 mM BME, 10 mM Imidazole, 1 tablet PI/L, pH 7.5Buffer B
50 mM HEPES, 0.3 M NaCl, 1 mM BME, 300 mM Imidazole, 1 tablet PI/L, pH 7.5Buffer C
50 mM HEPES, 0.3 M NaCl, 1 mM BME, pH 7.5Buffer D
50 mM HEPES, 50 mM NaCl, 1 mM DTT pH 7.2Buffer E
50 mM HEPES, 1 M NaCl, 1 mM DTT, pH 7.2