Selective leakage of host-cell proteins during high-cell-density cultivation of recombinant and non-recombinant Escherichia coli.
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.
Rinas
Ursula
U
Biochemical Engineering Division, GBF National Research Centre for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany. URI@gbf.de
Hoffmann
Frank
F
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
D029968:Escherichia coli Proteins; D011994:Recombinant Proteins