Strain: Hb101with Pks26r (Rho; Ampr); Pnt203 ( Ci857/ N; Tetr)

Protocol for overproduction and purification of Rho last updated 4/21/03 by Katsuya Shigesada

Strain HB101with pKS26R (rho; ampr); pNT203 (cI857/N; tetr) ref. pKS26R, Mori et al. JMB 210, 39-49, 1989; pNT203, Shigesada et al. Gene 29, 199-209, 1984; Tsurushita et al. JMB 210, 23-37, 1989. Upon arrival of an agar stub of this strain, please streak it immediately on an LB agar plate with ampicillin, 50 g/ml and 10 g/ml tetracycline, pick several well-grown colonies after overnight culture at 32 C, and make permanent freeze stocks from each of them after overnight culture in a few ml of LB as instructed below. In parallel, carry out heat induction in a small scale using residual portions of these cultures to check the rho-overproduction capacity. Keep those clones that pass this test. This is the surest way of generating and preserving good stocks of the super-efficient rho-overproducing strain. So I strongly recommend you always to adhere to the ritual.

Media Routine maintenance: LB (10g Bactotryptone, 5g Bacto yeast extract, 5g NaCl per liter) with ampicillin, 50 g/ml and 10 g/ml tetracycline.

Permanent freeze stock: a fresh overnight culture in the above medium is supplemented with 1/4 part(v/v) of 80%(v/v) glycerol, snap-frozen in 1-2 ml aliquots on liquid N2 or Dry-ice ethanol bath, and stored at –80 degree C or below. Preinduction culture: LB with ampicillin, 50 g/ml and 5 g/ml tetracycline

Induction: Maximum-induction medium (33g Bactotryptone, 20g Bacto yeast extract, 5g NaCl, 13.5g Na2HPO4.12H2O, 3g KH2PO4, 1g NH4Cl per liter) supplemented with 1ml 1M MgSO4, 10ml 10mM CaCl2, 5ml 20%(W/V) glucose per liter and 50 g/ml (final) ampicillin just before use (tetracycline should be omitted because it tends to inhibit rho overproduction as noted in Shigesada et al. Gene 29).

Procedures

1 Cell culture (1-liter scale affording purification of rho in a few hundred milligrams) 1. Using a small spatula, scoop a lump of frozen cells and dump them into 50-ml of preinduction medium. (Make this sampling on site as quickly as possible, so that the freeze stock should be kept frozen and then put the stock back to the freezer. This way, you can use the same stock many times until it runs out). 2. The culture is grown at 32 C overnight, and then combined with 1 liter of MIM medium containing only ampicillin, 50 g/ml. The culture is kept on at 32 C until the cell density reaches 100 Klett units (@ 1 OD at 660 nm). 3. The culture is quickly warmed up to 42 C by swirling on a 45 C water bath, and incubated at 42 C under vigorous shaking for another 3 hours. 4. Immediately after the termination of heat induction, 10 ml of 1 M MaN3 is added to the culture, which is then quickly chilled on ice and subjected to centrifugation at 9, 000 rpm for 10 min. The harvested cells are resuspended in 100 ml of ice-cold Lysis buffer and spun down in small centrifuge tubes at 15000 rpm for 10 min. Use preweighed bottles so as to estimate the net weight of the pelleted cells after centrifugation: about 10g of cells will be obtained usually. After this, the cells can be either processed immediately for purification, or stored frozen at –80 C until use.

Lysis buffer: 50 mM Tris.HCl (pH8.0), 5 mM EDTA, 0.25 M NaCl, 1 mM DTT and 30% (v/v) glycerol

Purification

Our procedure is an adaptation of the Finger & Richardson method with a few modifications: Finger LR, Richardson JP. Escherichia coli ribonucleic acid synthesis termination protein rho. Biochemistry. 20(6):1640-5, 1981.

Buffer G: 50 mM Tris.HCl, pH 8.0, 0.1 mM EDTA, 0.1 mM DTT and 20% (v/v) glycerol Polymin P working solution: 10% (w/v) Polymin P in distilled water, pH adjusted to 7.9 with 6N HCl (since 10% solution is too thick for direct pH measurement,

2 the amount of HCl to be added should be determined by pilot titration on 1% solution).

Step 1: preparation of crude cell extracts 1. Suspend the cells in 5 vol/wt of Lysis buffer kept ice-cold. To aid suspension, use a plastic pestle or a rubber policeman. When the suspension gets well-homogenized, PMSF is added to a final concentration of 0.1 mM (use 100 mM stock solution in isopropanol). 2. The cells are disrupted by sonication. (We used Branson, model 185E, in intermittent duty cycles (5 sec of irradiation followed by 10 sec of cooling off) for a total time of 10 min) 3. Add successively 1/50 vol of 1M MgCl2 and DNase I (RNase-free) to 15 g/ g cell (use 2 mg/l stock soln), and keep the suspension on ice for 60 min. 4. Centrifuge the suspension at 30, 000 rpm for 60 min. Collect the supernatant in a measuring cylinder and record the volume.

Step 2: Polymin P precipitation 1. Pilot titration: Dispense 100 l portions of the crude extracts into a series of microfuge tubes containing 0 to 20 l of Polymin P in 2-l incremental steps. Upon each addition, the content is quickly mixed, kept on ice for 10 min, and centrifuged for 5 min. The supernatants are diluted 50-fold in 50 mM Tris.HCl, pH 8.0 and scanned for UV spectrum (220-320nm). Plot the absorbance at 260 nm against the volume of Polymin P added, and find a point, X, where the initial steep down-ward slope abruptly shifts to a much gentler slope. An optimal v/v ratio of Polymin P to the extract is thus calculated to be X/100. 2. Add the calculated amount of Polymin P gradually to the bulk of crude extracts under gentle stirring, allow the precipitation reaction to complete for additional 30 min, and then centrifuge the mixture at 15, 000 rpm for 30 min. The supernatant is collected in a measuring cylinder and record its total volume.

Step 3: Ammonium sulfate precipitation 1. Add AmSO4 at a ratio of 3.9g/ml Polymin P supernatant under gentle stirring. For each 10g of AmSO4 added, 0.1 ml of 1N NaOH is added so as to prevent the mixture from becoming acidic. After all AmS04 is gone, the mixture is kept stirring for another 30 min and then centrifuged at 15, 000 rpm for 30 min. The precipitated proteins are redissolved in an

3 equal volume (ml) of buffer G per the weight (g) of the original cell pellet. The solution is desalted by dialysis against buffer G containing 0.05M NaCl or by passing through a small- pore gelfiltration column equilibrated with the same buffer.

Step 4: Chromatography on a S-Sepharose column 1. The desalted AmSO4 fraction is loaded onto a S-Sepharose column (bed volume: 50 ml) equilibrated with buffer G containing 0.05M NaCl. The column is washed with 200 ml of buffer G containing 0.05M NaCl, and then Rho is eluted with 250 ml of 0.05-0.5M NaCl gradient in buffer G. Rho is recovered in a single large peak centering at around 0.2M NaCl. Measure the conductivity of the pooled Rho fraction. If the conductivity is higher than that of 0.2M NaCl in buffer G, diluting the fraction with an appropriate amount buffer G containing no salt to make its salt concentration equivalent to 0.2M.

Step 5: Chromatography on a Heparin-Sepharose column 1. The S-Sepharose fraction is loaded onto a Heparin-Sepharose column (bed volume: 50 ml) equilibrated with buffer G containing 0.2M NaCl. The column is washed with 200 ml of buffer G containing 0.2M NaCl, and then Rho is eluted with 300 ml of 0.2-1M NaCl gradient in buffer G. Rho is recovered in a single large peak centering at around 0.6M NaCl. 2. The pooled Rho fraction is concentrated to a handy volume by an Amicon membrane concentrator, or any alternative one, if necessary. For a long-term storage, the final Rho prep is dialysed against buffer G containing 0.2M NaCl and 50% (v/v) glycerol, and kept unfrozen at –20 C.

That’s all. If a fast high-efficiency chromatography system is available, the whole purification procedure can be easily completed in two days. Good luck!