The following purification was implemented by Deb Taxman to purify GAGA factor expressed in E. coli.  The procedure is based on one originally provided by Toshio Tsukiyama when he was in Carl Wu's lab.  I have Toshio's original protocol in my files and I have incorporated some of his information.  pAR-GAGA is a plasmid originally prepared by Walter Soeller.  The first several amino acids have been altered.  We have another plasmid from Jim Kadonaga called pET-GAGA.  Deb Taxman's side by side comparison indicates pAT-GAGA expresses a few fold better than pET-GAGA.  Both plasmids can be found in my box labeled DJT.

Day 1.

Transform pAR-GAGA into competent BL21(pLysS) E. coli.  Plate on LB-Ampicillin/Chloramphenicol (Unfortunately, the concentration of Amp and Chloramphenicol were not given - Chwen-Huey has a PET vector expression manual that must have this information.  Also, chloramphenicol may not be essential as long as you are certain your cells are BL21(pLysS).  Toshio's protocol does not include it.
Toshio says the E.coli must be freshly transformed everytime you prepare GAGA factor!

Day 2.

Pick several colonies and inoculate 25 ml LB-Amp/Chloramphenicol.  Grow culture at 37oC for 3 hours (Preculture).

Inoculate 10 ml each of Preculture into two 4 liter flasks each containing 500 ml of LB-Amp.  Culture at 37oC until OD600=0.2.

Transfer flasks to 30oC shaker and continue to culture until OD600=0.6.  At this point, remove 1 ml of cells to serve as an uninduced control - place on ice.  Add IPTG to a final concentration of 0.4 mM and continue to grow cells for 30 minutes.

Thirty minutes after adding IPTG, Deb Taxman added rifampicin from a 20 mg/ml stock to give a final concentration of 40 ug/ml.  The cells were incubated for an additional 30 minutes.  This may have increased the yield of protein but the effect was not very substantial.  Toshio did not add rifiampicin and collected cells 30 minutes after adding IPTG.

Remove 1 ml of cells to compare with uninduced cells - place on ice.

Collect remaining cells by centrifuging at 5000 rpm for 20 minutes in the GS3 rotor at 4oC.

Suspend cells in 100 ml of ice cold PBS. Then collect cells by centrifuging 5000 rpm for 20 minutes.

Remove the supernatant and store the cell pellet at -75oC.

Collect the cells from the 1ml aliquots of uninduced and induced cells by centrifuging the samples for 1 minute in a microfuge.  Dissolve the cells in 100 ul of SDS-PAGE sample buffer.  Boil and analyze on a 8% acrylamide gel.  GAGA factor should be visible in the induced sample just above where the BSA marker runs.

Day 3 (or sometime after you have run the SDS-PAGE to ascertain that GAGA factor has been expressed.

Equilibrate a 10 ml DEAE sepharose column with 0.3 M HEMGN (25 mM Hepes-KOH pH 7.6, 0.1 mM EDTA, 12.5 mM MgCl2, 10% glycerol, 0.1% NP40, 1 mM DTT, 0.1 mM PMSF plus 0.3 M KCl).  Also equilibrate a Heparin Sepharose CL-6B column with 0.3 M HEMGN.  Unfortuantely, neither Toshio nor Deb indicate in their protocols how large this column should be.  I suspect that it must be around 5 mls because Deb appears to have collected 2.5 ml fractions.  Check out Tsukiyama and Wu (1995) Cell 83:1011-1020 for more details since this is where the purification was published.

Resuspend cells in 10 to 20 ml of 0.3 M HEMGN and sonicate with 15 second pulses set to give maximum cavitation.  Sonicate 4 times.  Toshio indicated that the OD600 of the lysate should be less than 10% of the original so this implies to me that he must have measured some of the cell suspension before doing the sonication (probably measured a dilutions since the cells have been concentrated 100 fold from the original culture).

Centrifuge the lysate at 10K for 10 minutes.

Load supernatant onto a 10 mL DEAE Sepharose.  Collect the flow through and subsequent 20 ml of wash solution.  GAGA factor is in the flowthrough.

Load flowthrough and wash onto Heparin Sepharose and wash with five column volumes of 0.3M HEMGN.  Elute protein with steps of 0.4M, 0.5M and 1M HEMGN.  Each step should be with 4 column volumes and collect fractions that are 1/2 a column volume.

Analyze fractions by SDS-PAGE - GAGA should be mostly in the 0.5 M fraction and is 80% pure.  This is adequate for most work.

Toshio indicates that further purification can be achieved by dialyzing the fractions to 0.1 M HEMGN and chromatographing the material on Mono S.  The column is washed at 0.2 M HEMGN and GAGA factor is eluted with 0.4 M HEMGN.