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).

i.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)

ii.Grow for 6 to 8 hours at 37 ℃

iii.Inoculate 0.2 ml of culture into 25 ml of LB containing Kanamycin and grow at 37 ℃,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)

iv.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 ℃ until the cells reach an OD₆₀₀ of 0.8.

v.Induce cells with IPTG to a final concentration of 1 mM and grow for another 3 to 4 hours at 37 ℃.

vi.Harvest the bacteria by centrifugation at 7000 rpm (20 min)and freeze the cells at -20 ℃ for lysis/purification the following day.

vii.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.

viii.Incubate the cells ice for 10 min and add NP-40 and MgCl₂ to a final concentration of 1% and 1 mM,respectively.

ix.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

x.Centrifuge the E.Coli extract at 22,000 rpm for 30 min

xi.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 ℃ for 10 min.Spin down at 22,000 rpm for 20 min to remove the precipitated nucleic acids.

xii.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)

xiii.Use a gradient program in FPLC or AKTA to purify the fusion protein.Set the gradient to be at least 2 column volumes

xiv.After collecting fractions clean the column with 1 column volume of Buffer B followed by storage with 20% EtOH.

xv.Pool fractions (judged from SDS-PAGE analysis)and add thrombin (200U for 6 L prep.)and dialyze sample against buffer CO/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 100μl of thrombin.

xvi.Pass the sample over pre-equilibrated benzamidine and Ni-NTA beads (set up gravity column)

xvii.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 CO/N and proceed for MonoS column purification.

xviii.Clean the monoS column with 4 column volumes of Buffer E and equilibrate with 4 column volumes of Buffer D

xix.Load the fractions using super loop and set-up a gradient program for 10 column size

xx.The protein elutes as a single sharp peak.Collect the fraction and freeze the protein at -80 ℃ 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