PURIFICATION OF RPA
The paper describing the initial characterization of recombinant RPA (Henricksen et al, JBC 269, 11121) also describes the general protocol for induction and purification. Of the two purification procedures described in the paper, we always use hydroxylapatite instead of dialysis. This is easier and gives better yields. This purification is also described in detail in Binz, S.K., Dickson, A.M., Haring, S.J., and Wold, M.S. (2006) "Functional assays for replication protein A (RPA)." Meth. of Enzymol. Volume 409, DNA Repair, Part B, J.L. Campbell & P. Modrich, Edit., pp11-38.
HI Buffer: 30 mM HEPES (from a 1 M stock at pH 7.8), 0.25 mM EDTA, 0.25% (w/v) myo‐inositol, 1 mM dithiothreitol (DTT), and 0.01% (v/v) Nonidet‐P40. The inositol and detergent act as protein stabilizers and can be replaced by 10% glycerol and other non‐ionic detergents (e.g., Tween‐20), respectively. DTT should be added right before using the buffer (it oxidizes over time and is no longer effective after about 24 hours). HI-X Buffer - X mM indicated salt).
Induction and lysis.
We have found that RPA is toxic to E. coli so we always try to minimize the number of generations (and time in stationary phase) between transformation and induction. Thus we induce from a fresh transformant of p11d-tRPA (preferably 1-2 days after transformation) and grow the cells as described in the paper. Briefly: Inoculate 1 liter of TB-Amp in a 2.8 liter fernbach flask with a single colony. Place at 25° or 37°C WITHOUT shaking overnight. Generally, the culture will reach a OD600 ~ 0.1-.3 after growing overnight. The next morning, place the culture on the shaker and begin agitation at ~ 200 - 250 rpm. Grow at 37°C with shaking until OD600 = 0.5 - 0.8 (generally, 2-5 hr depending on the culture). Induce expression of protein by adding IPTG to a final concentration of 0.3 mM IPTG (3 ml of 100 mM IPTG). The cells are pelleted by centrifugation at 4000 rpm at 4°, then resuspended in HI buffer containing final concentrations of 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM DTT, and 2 l/ml of bacterial protease inhibitor cocktail (Sigma, catalogue number P8465, St. Louis, MO). Cells can either be frozen at -80° or lysed. We usually lyse the cells using a french press or cell breaker. (We, and others, have also had success using sonication to lyse the cells.)
Outline of purification steps.
1. Soluble lysate from an induced culture is applied an Affi-Gel Blue (Bio-Rad) column equilibrated with HI buffer containing 50 mM KCl (load at 8-10 mg protein/ml resin). The column was washed sequentially with 3 column volumes of HI buffer containing 50 mM KCl, 0.8 M KCl, 0.5 M NaSCN or 1.5 M NaSCN. RPA elutes in the 1.5 M NaSCN wash.
2. The peak of protein from the 1.5M NaSCN wash is applied directly to a HAP column equilibrated with HI buffer with 30 mM KCl. We load at 5 mg protein per ml of resin and generally load at the fastest flow rate practical. (This can be done by gravity flow or on an automated chromatography apparatus.) The RPA fractions can be frozen between the Affi-gel blue and HAP columns but we find that we get better recovery if the fractions are run over HAP as quickly as possible. The HAP column is washed sequentially with 3 column volumes each of HI buffer containing 0, 80 or 500 mM potassium phosphate. RPA generally elutes in 80 mM phosphate. However, in some preparations the RPA elutes in the 0 mM wash. This does not seem to any a difference in the final yield or activity but the peak contains some NaSCN so it will have to be diluted more extensively before loading onto Mono-Q. After this step the RPA should be 80-90+% pure. Lower purity usually indicates poor induction/low RPA levels in lysate.
3. 80 mM phosphate fraction is then diluted 2-4 fold into HI buffer with 0 salt (to reduce ionic strength below 100 mM and applied to a Mono-Q (HR5/5) column equilibrated with HI buffer containing 50 mM KCl. (We load up to 8 mg protein onto this column. Larger amounts of protein are either split into two column runs or loaded onto a bigger Mono-Q column.) The column is washed sequentially with 4 ml HI buffer containing 50 mM KCl and 100 mM KCl. (We sometimes skip the 50 mM wash.) The column is eluted with a 10 ml gradient HI buffer with from 200-400 mM KCl. RPA elutes ~300 mM. At this point RPA should be 95+% pure. In some preps there is a 71 kDa E. coli protein that elutes close to RPA in this step. Usually we get ~50% recovery of the RPA on this column. Occasionally we see a much lower recovery, <10%. We believe this is due to protein aggregation but do not know what causes this to happen.
4. The peak from Mono-Q should be 95+% pure and be stable through multiple freeze/thaw cycles and for long periods at -80°C.
Alternate Buffers: J Buffer (for improved RPA stability)
We have recently started using an alternate buffer that seems to minimize RPA aggregation and improves RPA stability.
1X J-0 Buffer: 30 mM Hepes, 0.5% inositol, 0.25 mM EDTA, 1 mM tris(2-carboxyethyl)phosphine, 0.02% Tween-20).
J-0 Stock 500 ml 1 L 2 L
1 M Hepes, pH 7.8 15 ml 30 ml 60 ml
10 % Inositol 25 ml 50 ml 100 ml
0.5 EDTA 0.25 ml 0.5 ml 1 ml
10 % Tween 20 1 ml 2 ml 4 ml
1 M TCEP 0.5 ml 1 ml 2 ml
• TCEP provides selective reducing potential for disulfide bonds and does not react with oxygen. Thus, it is stable for extended periods of time. The exception is that TCEP reacts with phosphate and should be added immediately before use with phosphate buffers.
• After adding salt (if needed) increase volume with ddH2O and filter.