title: Selective leakage of host-cell proteins during high-cell-density cultivation of recombinant and non-recombinant Escherichia coli.
abstract: Significant leakage of host-cell proteins into the culture medium occurred during high-cell-density cultivation of E. coli. Identification of these medium proteins revealed almost exclusively a periplasmic origin. Release of periplasmic proteins into the culture medium was observed throughout the entire cultivation of recombinant or non-recombinant cells. Leakage was intensified, however, in the final part of high-cell-density cultures (>100 g L(-)(1) dry cell mass) or when a temperature upshift was used for induction of recombinant protein synthesis. After temperature upshift, formation rates and residual cellular concentrations of periplasmic proteins declined with individual rates; e.g., the cellular content of the large periplasmic dipeptide binding protein DppA (57.4 kDa) started to decline about 4 h after the temperature upshift, whereas the smaller periplasmic d-galactose/d-glucose binding protein MglB (33.4 kDa) was already lost during the first hour after the upshift. In addition to periplasmic proteins, the osmotic-shock-sensitive heat-shock protein DnaK was found in significantly higher proportion in the cell-free medium of the temperature-challenged culture than other cytoplasmic proteins. Cell lysis was not observed even after prolonged cultivation. Thus, loss of a subset of cellular proteins of mainly periplasmic origin ordinarily occurs during cultivation and is intensified through stressful conditions in high-cell-density cultures. The selective release of cellular proteins of periplasmic origin offers the opportunity to simplify the downstream processing of recombinant proteins directed to the periplasm of E. coli.
authors.affiliation: Biochemical Engineering Division, GBF National Research Centre for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany. URI@gbf.de
authors.forename: Ursula
authors.initials: U
authors.lastname: Rinas
authors.forename: Frank
authors.initials: F
authors.lastname: Hoffmann
mesh_terms: D019149:Bioreactors / Q000382:microbiology; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D018384:Oxidative Stress / Q000502:physiology; D019897:Periplasm / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000378:metabolism*; D013696:Temperature
chemical_list: D029968:Escherichia coli Proteins; D011994:Recombinant Proteins