Details
| Original language | English |
|---|---|
| Pages (from-to) | 61-67 |
| Number of pages | 7 |
| Journal | Biotechnology and bioengineering |
| Volume | 66 |
| Issue number | 1 |
| Publication status | Published - 26 Mar 2000 |
| Externally published | Yes |
Abstract
The synthesis of a proteolytically unstable protein, originally designed for periplasmic export in recombinant Escherichia coli BL21(DE3), a strain naturally deficient for the ATP-dependent protease Lon (or La) and the outer membrane protease OmpT, is associated with a severe growth inhibition. This inhibition is not observed in BL21(DE3) synthesizing a closely related but proteolytically stable protein that is sequestered into inclusion bodies. It is shown that the growth inhibition is mainly caused by a slower cell division rate and a reduced growth yield and not by a general loss of cell division competence. Cells proceed with their normal growth characteristics when exposed again to conditions that do not sustain the expression of the heterologous gene. The performance of cells synthesizing either the stable or the degraded protein was also studied in high cell density cultures by employing a new method to calculate the actual specific growth rate, the biomass yield coefficient, and the dissimilated fraction of the carbon substrate in real-time. It is shown that the growth inhibition of cells synthesizing the proteolytically degraded protein is connected to an increased dissimilation of the carbon substrate resulting in a concomitant reduction of the growth rate and the biomass yield coefficient with respect to the carbon source. It is postulated that the increased dissimilation of the carbon substrate by Ion-deficient BI21(DE3) cells synthesizing the proteolytically unstable protein may result from a higher energy demand required for the in vivo degradation of this protein by ATP-dependent proteases different from the protease Lon.
Keywords
- Carbon metabolism, Proteolytic degradation, Recombinant protein, Stress response
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Biotechnology
- Chemical Engineering(all)
- Bioengineering
- Immunology and Microbiology(all)
- Applied Microbiology and Biotechnology
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In: Biotechnology and bioengineering, Vol. 66, No. 1, 26.03.2000, p. 61-67.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Secretion-dependent proteolysis of heterologous protein by recombinant Escherichia coli is connected to an increased activity of the energy- generating dissimilatory pathway
AU - Schmidt, Michael
AU - Viaplana, Elisenda
AU - Hoffmann, Frank
AU - Marten, Sabine
AU - Villaverde, Antonio
AU - Rinas, Ursula
PY - 2000/3/26
Y1 - 2000/3/26
N2 - The synthesis of a proteolytically unstable protein, originally designed for periplasmic export in recombinant Escherichia coli BL21(DE3), a strain naturally deficient for the ATP-dependent protease Lon (or La) and the outer membrane protease OmpT, is associated with a severe growth inhibition. This inhibition is not observed in BL21(DE3) synthesizing a closely related but proteolytically stable protein that is sequestered into inclusion bodies. It is shown that the growth inhibition is mainly caused by a slower cell division rate and a reduced growth yield and not by a general loss of cell division competence. Cells proceed with their normal growth characteristics when exposed again to conditions that do not sustain the expression of the heterologous gene. The performance of cells synthesizing either the stable or the degraded protein was also studied in high cell density cultures by employing a new method to calculate the actual specific growth rate, the biomass yield coefficient, and the dissimilated fraction of the carbon substrate in real-time. It is shown that the growth inhibition of cells synthesizing the proteolytically degraded protein is connected to an increased dissimilation of the carbon substrate resulting in a concomitant reduction of the growth rate and the biomass yield coefficient with respect to the carbon source. It is postulated that the increased dissimilation of the carbon substrate by Ion-deficient BI21(DE3) cells synthesizing the proteolytically unstable protein may result from a higher energy demand required for the in vivo degradation of this protein by ATP-dependent proteases different from the protease Lon.
AB - The synthesis of a proteolytically unstable protein, originally designed for periplasmic export in recombinant Escherichia coli BL21(DE3), a strain naturally deficient for the ATP-dependent protease Lon (or La) and the outer membrane protease OmpT, is associated with a severe growth inhibition. This inhibition is not observed in BL21(DE3) synthesizing a closely related but proteolytically stable protein that is sequestered into inclusion bodies. It is shown that the growth inhibition is mainly caused by a slower cell division rate and a reduced growth yield and not by a general loss of cell division competence. Cells proceed with their normal growth characteristics when exposed again to conditions that do not sustain the expression of the heterologous gene. The performance of cells synthesizing either the stable or the degraded protein was also studied in high cell density cultures by employing a new method to calculate the actual specific growth rate, the biomass yield coefficient, and the dissimilated fraction of the carbon substrate in real-time. It is shown that the growth inhibition of cells synthesizing the proteolytically degraded protein is connected to an increased dissimilation of the carbon substrate resulting in a concomitant reduction of the growth rate and the biomass yield coefficient with respect to the carbon source. It is postulated that the increased dissimilation of the carbon substrate by Ion-deficient BI21(DE3) cells synthesizing the proteolytically unstable protein may result from a higher energy demand required for the in vivo degradation of this protein by ATP-dependent proteases different from the protease Lon.
KW - Carbon metabolism
KW - Proteolytic degradation
KW - Recombinant protein
KW - Stress response
UR - http://www.scopus.com/inward/record.url?scp=0033230145&partnerID=8YFLogxK
U2 - 10.1002/(SICI)1097-0290(1999)66:1<61::AID-BIT6>3.0.CO;2-G
DO - 10.1002/(SICI)1097-0290(1999)66:1<61::AID-BIT6>3.0.CO;2-G
M3 - Article
C2 - 10556795
AN - SCOPUS:0033230145
VL - 66
SP - 61
EP - 67
JO - Biotechnology and bioengineering
JF - Biotechnology and bioengineering
SN - 0006-3592
IS - 1
ER -