MATERIALS AND METHODS. Protein Cloning and Purification

RecBCD was purified from E. coli JM109 co-transformed with plasmids pPB800 and pPB520, which were a generous gift from Dr. Jeff Gelles (Brandeis University) 1,2. Cells were grown in 2YT in the presence of 34 µg ml-1 chloramphenicol and 50 µg ml-1 carbenicillin. The cells were grown to an OD600 ~0.6, induced with IPTG, collected 4 hours after induction, resuspended in buffer R (50 mM Tris-HCl pH 7.5, 0.1 mM PMSF, 10 % sucrose), and lysed by freezing and sonication 1. The lysate was clarified by high-speed centrifugation and fractionated with 0.282 g ml-1 ammonium sulfate. The precipitated protein was recovered by centrifugation at 20,000g for 20 min. The pellet was resuspended in buffer A (20 mM Tris-HCl [pH 7.5], 0.1 mM DTT, 0.1 mM EDTA) and loaded onto a 5 ml HiTrap Q FF column (GE Healthcare). The protein was eluted with a gradient to 100 % buffer B (20 mM Tris-HCl [pH 7.5], 0.1 mM DTT, 0.1 mM EDTA, 1 M NaCl). The protein-containing fraction were diluted with buffer A to a final NaCl concentration of less than 0.1 M, and loaded onto a HiPrep Heparin 16/10 FF column (GE Healthcare). The protein was eluted with a gradient to 50 % buffer B, loaded directly onto a Mono Q 5/50 GL column, and eluted with a gradient to 100 % buffer B. The pooled protein fractions were loaded onto a HiPrep 16/60 Sephacryl S-300 HR column (GE Healthcare). Followin