T7 RNAP Protein Prep
- prepare (4) 1L- LB solutions and sterilize
I. Cell Growth
-prepare 1000x Ampicillin solution* ( *= see notes pg.)
-add 1ml of the 1000x Amp solution to each LB flask
-either transfer one colony or a scraping from a glycerol stock of BL21-pAR1219 to a 6ml solution of
LB/Amp(100M/ml) and incubate at 37C with shaking ~220 rpm, until A600 reaches 0.5 (make one
6ml solution for each flask that you intend to grow)
-pour the 6ml tube of cells into one flask and incubate at 37C with shaking until A600=0.5
-add 1ml of 1000x IPTG* to each flask to induce T7 RNAP production and continue incubation for
4hrs
-centrifuge (4,200 rpm for 20min), then wash cells twice with 100ml Wash Buffer* (pellets can be
stored at -80C) [obtain ~6-8g/L]
-run 10% SDS PAGE in Tris Glycine (SDS gel loading dye, constant current 20mA, stained in
Coomassie Blue) to check expression of RNAP in the cells
II. Cell Lysis
-thaw cells in ice bath, then suspend in 70ml cold Buffer LB*
-initiate lysis by adding 17ml of a fresh solution of egg white lysozyme (in buffer LB)*, also add
150l PMSF* and 70l leupeptin*
[although the cell line BL21 lacks the major protease that cuts T7 RNAP, the enzyme is very
sensitive to proteolysis]
-let stand 20min
-add 9ml of 0.8% sodium deoxycholate* and allow to stand for another 20min to complete lysis [The
solution will become highly viscous. To reduce viscosity sonicate for ~15sec 5xs]
-add 150l PMSF
-add 17ml 2M Ammonium sulfate* to the lysate
-add Buffer LB to bring the solution up to 170ml if needed
-add 17ml of 10% PEI* slowly with vigorous stirring on ice
-after 20min remove the precipitate by centrifugation (39,000g 15min)
-the supernatant, which contains the RNAP, is slowly mixed on ice with 0.82 volumes of a saturated
solution of enzyme grade ammonium sulfate (a saturated solution at room temperature was adjusted
to pH 7 by adding Tris base, then filtered and chilled to 4C prior to use)
[DO NOT add fast as it can cause degradation of the polymerase]
-stir for 15-20min on ice
-collect the precipitate by centrifugation (12,000g 10min)
-dissolve the precipitate in 50ml of Buffer C* + 100mM NaCl
-this fraction is dialyzed O/N against 4L of Buffer C + 100mM NaCl, also add 4ml PMSF to dialysis
III. Purification
-pack first column with pre-swollen degassed sephadex SP (C-50) resin [autoclaved 6g resin in
ddH2O], then equilibrate column with Buffer C + 50mM NaCl
-centrifuge dialyzed protein (12,000g 20min)
-take the conductivity of Buffer C + 50mM NaCl and of supernatant. Dilute the supernatant with
Buffer C until its conductivity is equal to that of Buffer C + 50mM NaCl. Also check the conductivity
of the column wash.
-add 150l PMSF to the supernatant
-load the diluted solution onto the column and wash the column with 3.5x the volume of the column
with Buffer C + 50mM NaCl
-after all peaks come off of the column, elute the RNAP with Buffer C + 200mM NaCl that contains
1ml PMSF
-after the elution, run an SDS-PAGE to confirm the protein
-dialyze the protein in 3L of Buffer C + 25mM NaCl
-make the conductivity of the dialyzed solution equal to that of Buffer C + 25mM NaCl with Buffer C
without salt
-column two was packed with DEAE resin and equilibrated with Buffer C + 25mM NaCl
-load the protein onto the column, then wash with Buffer C + 25mM NaCl
-elute the DEAE column with a gradient of Buffer C + 25-250mM NaCl [peak should begin around
100-120mM NaCl]
-run an SDS-gel to locate the purest fractions of RNAP
-pool the purest fractions and load into a amicon concentrator containing a YM30 ultrafiltration
membrane (membrane had been soaked in ddH2O shiny side down at least an hour, then placed shiny
side up in the amicon). The concentrator was attached to a Nitrogen tank under ~30 psi.
-dialyze the protein against 2L Buffer C + 100mM NaCl + 50% glycerol O/N
-measure protein concentration at A280 using an extinction coefficient of 1.4 x 105.
-aliquot the protein and store at -80C
IMPORTANT!!!!->perform an exonuclease assay. If the first column if overloaded an exonuclease
may remain with the polymerase
-the DEAE column can be recycled by washing with 400mM NaCl for 4 column volumes then
washing with 20% ethanol
reference: Grodberg & Dunn, J. of Bacteriology. vol 170, pg1245-1253, 1988
We follow their guidelines with the exception that we do no use a sephadex CM column after
the SP column and before the DEAE column.
Notes Page
LB media = for 1L, 10g tryptone, 5g yeast extract, 10g NaCl. bring volume to 1L then sterilize
1000x Ampicillin Solution
1x Amp=100/ml; 1000x=100mg/ml so dissolve 1g Amp in 10ml ddH2O
1000x IPTG
IPTG FW=238.3g; 0.5M=119.15mg/1ml ddH2O = 1000x
Wash Buffer = [20mM TrisHCl (pH 8.1), 20mM NaCl, 2mM EDTA]
Buffer LB = [50mM TrisHCl (pH 8.1), 20mM NaCl, 2mM EDTA, 1mM DTT]
egg white lysozyme = 1.5mg/ml; dissolve 108mg lysozyme in 72ml Buffer LB
PMSF = 20mg/ml; dissolve 200mg in 10ml isopropanol; MUST be made fresh each time used
leupeptin = 5mg/ml; dissolve 1mg in 200l ddH2O
0.8% sodium deoxycholate = dissolve 200mg in 25ml ddH2O
Ammonium sulfate; FW=132; 2M Am.sulfate = 13.2g in 50ml ddH2O
10% PEI = made 25ml by adding 2.5ml PEI and some water in a 50ml beaker, adjust to pH 8 with
concentrated HCl, then make up to 25ml
Buffer C = 20mM sodium phosphate (pH 7.7), 1mM EDTA, 1mM DTT, 5% glycerol
[it is useful to make a stock of 10x buffer C]
10x buffer C= 62.65g Na2HPO4
3.45g NaH2PO4
20ml 0.5M EDTA
for 1 L (without glycerol or DTT)