Rapid and Efficient Extraction of Soluble Proteins from Gram-negative Microorganisms without Disruption of Cell Walls
V. N. Danilevich#, L. E. Petrovskaya, and E. V. Grishin
#Phone: (495) 336-65-40; fax: (495) 330-73-01; e-mail: email@example.com Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117997, Russia Received: February 17, 2006; in final form: March 31, 2006
Abstract: The ability of buffer solutions containing low concentrations of nonionic detergents (Triton X-100, Tween 20, Brij 58, and Lubrol PX) and the anionic detergent sodium deoxycholate, as well as mixtures of these detergents with chaeotropes (urea and guanidine hydrochloride), to extract intracellular proteins of Gram-negative microorganisms (Escherichia coli and Pseudomonas aeruginosa) was studied. It was established that the solutions containing Triton X-100 and sodium deoxycholate and the mixtures of these detergents with urea are the most effective. It was shown that the extraction of proteins from bacterial cells under the studied conditions is not accompanied by a release of DNA into solution but is associated with extraction of low-molecular RNAs. The level of protein extraction reaches 80%. No disruption of the bacterial cell wall occurs during the extraction, and proteins probably penetrate through meshes of the murein network. The efficiencies of our buffer mixtures are close to or higher than that of the commercial reagent CelLytic B (Sigma, United States). The practical uses of the chaeotropic mixtures developed are discussed.
Key words: Escherichia coli, extraction of proteins and RNAs, Gram-negative microorganisms, nonionic and anionic detergents, mild chaeotropic solutions, SDS-PAGE
Russian Journal of Bioorganic Chemistry 2006, 32 (6): 521-528