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A microscale method of protein extraction from bacteria: Interaction of Escherichia coli with cationic microparticles

Author:
Trefilov, Alexandru, Imendoerffer, Moritz, Sekot, Gerhard, Strobl, Florian, Jungbauer, Alois, Hahn, Rainer
Source:
Journal of biotechnology 2015 v.207 pp. 21-29
ISSN:
0168-1656
Subject:
DNA, Escherichia coli, adsorption, anion exchange, anions, bacteria, cell membranes, cell walls, desorption, endotoxins, flocculation, homogenization, outer membrane proteins, recombinant proteins, sodium chloride
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.
Agid:
5521215