title: A microscale method of protein extraction from bacteria: Interaction of Escherichia coli with cationic microparticles.
abstract: We developed a simple, highly selective, efficient method for extracting recombinant proteins from Escherichia coli. Our recombinant protein yield was equivalent to those obtained with high pressure homogenization, and did not require exposure to harsh thermal, chemical, or other potentially denaturing factors. We first ground conventional resin, designed for the exchange of small anions, into microparticles about 1μm in size. Then, these cationic microparticles were brought convectively into close contact with bacteria, and cell membranes were rapidly perforated, but solid cell structures were not disrupted. The released soluble components were adsorbed onto the cell wall associated microparticles or diffused directly into the supernatant. Consequently, the selective adsorption and desorption of acidic molecules is built into our extraction method, and replaces the equally effective subsequent capture on anion exchange media. Simultaneously to cell perforation flocculation was induced by the microparticles facilitating separation of cells yet after desorption of proteins with NaCl. Relative to high pressure homogenization, endogenous component release was reduced by up to three orders of magnitude, including DNA, endotoxins, and host cell proteins, particularly outer membrane protein, which indicates the presence of cell debris.
authors.affiliation: Austrian Centre of Industrial Biotechnology, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Alexandru
authors.initials: A
authors.lastname: Trefilov
authors.affiliation: Austrian Centre of Industrial Biotechnology, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Moritz
authors.initials: M
authors.lastname: Imendoerffer
authors.affiliation: Austrian Centre of Industrial Biotechnology, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Sekot
authors.affiliation: Austrian Centre of Industrial Biotechnology, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Florian
authors.initials: F
authors.lastname: Strobl
authors.affiliation: Austrian Centre of Industrial Biotechnology, Muthgasse 18, 1190 Vienna, Austria; Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna (BOKU), Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Alois
authors.initials: A
authors.lastname: Jungbauer
authors.affiliation: Austrian Centre of Industrial Biotechnology, Muthgasse 18, 1190 Vienna, Austria; Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna (BOKU), Muthgasse 18, 1190 Vienna, Austria. Electronic address: Rainer.Hahn@boku.ac.at.
authors.forename: Rainer
authors.initials: R
authors.lastname: Hahn
mesh_terms: D000327:Adsorption; D002412:Cations; D002473:Cell Wall / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005427:Flocculation; D053758:Nanoparticles / Q000737:chemistry*; D010316:Particle Size; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification*
chemical_list: D002412:Cations; D011994:Recombinant Proteins
keywords: Cell debris; Cell disruption; Cell membrane perforation; DNA; Endotoxin; Floc formation; High pressure homogenization; Host cell protein; Ion exchange; Micro scale process; Microparticles; Protein extraction