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

5 -measure protein concentration at A280 using an extinction coefficient of 1.4 x 10 . -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)