Compaction Agent Protection of Nucleic Acids during Mechanical Lysis Jason C. Murphy, †,§ Tony Cano, †,| George E. Fox, ‡ and Richard C. Willson* ,†,‡ Department of Chemical Engineering and Department of Biology and Biochemistry, University of Houston, 4800 Calhoun Avenue, Houston, Texas 77204 Mechanical lysis is an efficient and widely used method of liberating the contents of microbial cells, but the sensitivity of large nucleic acids to shear damage has prevented the application of mechanical lysis to DNA purification. It is demonstrated that polycationic compaction agents can protect DNA from shear damage and allow chromosomal and plasmid DNA purification by mechanical lysis. In addition to being substantially protected during mechanical lysis, the compacted DNA can be separated with the insoluble cell debris, washed, and selectively resolubilized, yielding a substantially purified DNA product. An additional benefit of this method is that lysate viscosity is greatly reduced, allowing the use of much smaller processing volumes when compared with traditional lysis methods used in nucleic acid purification. Introduction With numerous DNA vaccines and gene therapy products now in clinical trials, there is considerable demand for improved large-scale DNA separation techniques (1-3). One major area of concern during large-scale DNA production is the initial cell lysis step (4-6). Current lysis techniques for DNA production require caustic solutions, enzymes, and/or heat to liberate DNA from bacterial cells (7-9). In addition, each of these lysis methods requires the use of large volumes to keep viscosity within manageable limits. Intracellular proteins have long been recovered by mechanical lysis methods including homogenization, bead milling, and sonication. These induce lysis by shear, impingement, cavitation, pressure shock, and combinations of these mechanisms (10- 12). Large nucleic acids such as plasmid and chromosomal DNA, however, are mechanically sheared and fragmented by standard mechanical lysis techniques at strain rates of ap- proximately 10 5 -10 6 s -1 (13-15). We have previously shown that condensation/compaction of DNA can enhance its adsorption on chromatographic media at low ionic strength (16) and can serve as the basis for selective precipitation and fractionation of DNA and RNA (17-19). Compaction employs synthetic versions of small, natural poly- cations such as spermine and spermidine (20, 21) to induce reversible conformational changes and/or precipitation of nucleic acids. This technique has been shown to produce highly purified DNA with high efficiency (22). Compaction agents can also be used to eliminate nucleic acids from lysates containing desired nonnucleic acid products (e.g., proteins and polysac- charides), reducing lysate viscosity and eliminating the need for nucleases or additional nucleic acid removal steps (23). It is shown herein that compaction-agent-induced protection by condensation of plasmid and chromosomal DNA enhances plasmid DNA yields after mechanical lysis; the precipitated DNA can readily be selectively redissolved away from insoluble cell debris. Experimental Methods E. coli strain JM109 containing plasmid pCMV sport  gal (Gibco BRL) was grown in a 20-L Applikon fermenter, harvested, pelleted by centrifugation, and stored at -80 °C. “Compaction protection buffer” consisting of 0.5% w/v Brij 58 with added spermidine (15-30 mM) in 20 mM Tris HCl at pH 8.0 was added directly to frozen cell mass at 7-10 mL per gram of wet cells (100-150 mL total volume per French cell press cycle, resulting optical density ca. 80-100). Control experiments were also conducted with a buffer containing 0.5% w/v Brij 58 in 20 mM Tris HCl at pH 8.0 (no spermidine). The nonionic detergent Triton X-100 (1% w/v) performed equiva- lently to 0.5% w/v Brij 58. The mixture was vortexed to disperse the cells, allowed to stand for 4 min, and mechanically lysed using a French press (PC-160, SLM Aminco) at a pressure of 4,000-12,000 psig (vide infra). The lysed cells were then further processed by one of two alternative methods. A. Protected Lysis Only. The initial approach was to add 1 volume of 1.2 M NaCl in 20 mM Tris HCl, pH 8.0 to the spermidine-protected lysate in order to resuspend the plasmid into the supernatant while leaving the majority of cell contents insoluble. The mixture was centrifuged (10,000 × g, 15 min), and the supernatant was decanted. For electrophoresis, the supernatant was desalted by addition of 0.7 volumes of 2-propanol, 1-h incubation at -20 °C, and centrifugation at 10,000 × g for 15 min. The resulting pellet was then resuspended in TAE (40 mM Tris-HCl, pH 8.0, 40 mM acetic acid, and 1 mM EDTA) and analyzed by gel electrophoresis. B. Protected Lysis with Further Separation. In the second approach, the lysate was centrifuged at 10,000 × g for 15 min (with the DNA still compacted and insoluble). The supernatant was discarded, and the compaction-precipitated DNA/cell debris pellet was mixed with stripping solution (50% ethanol, 600 mM NaCl with 10 mM EDTA; 1 mL per 10 mL initial lysate) to remove the spermidine from the pellet while keeping the DNA * To whom correspondence should be addressed. Ph: (713) 743-4308. Fax: (713) 743-4323. E-mail: willson@uh.edu. † Department of Chemical Engineering. ‡ Department of Biology and Biochemistry. § Present address: Merck Research Laboratories, P.O. Box 4, West Point, Pennsylvania 19486-0004. | Present address: Genentech, Inc., 1 DNA Way, South San Francisco, California 94080. 519 Biotechnol. Prog. 2006, 22, 519-522 10.1021/bp050422i CCC: $33.50 © 2006 American Chemical Society and American Institute of Chemical Engineers Published on Web 03/15/2006