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Biotechnol Prog. 2004 May-Jun;20(3):679-87.

Selective leakage of host-cell proteins during high-cell-density cultivation of recombinant and non-recombinant Escherichia coli.

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1
Biochemical Engineering Division, GBF National Research Centre for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany. URI@gbf.de

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.

PMID:
15176868
DOI:
10.1021/bp034348k
[Indexed for MEDLINE]
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