Monitoring of genes that respond to overproduction of an insoluble recombinant protein in Escherichia coli glucose-limited fed-batch fermentations.
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Monitoring of genes that respond to overproduction of an insoluble recombinant protein in Escherichia coli glucose-limited fed-batch fermentations. / Jürgen, B; Lin, Hongying; Riemschneider, S; Scharf, C; Neubauer, P; Schmid, R; Hecker, M; Schweder, T.
In: BIOTECHNOL BIOENG, Vol. 70, No. 2, 2, 2000, p. 217-224.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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TY - JOUR
T1 - Monitoring of genes that respond to overproduction of an insoluble recombinant protein in Escherichia coli glucose-limited fed-batch fermentations.
AU - Jürgen, B
AU - Lin, Hongying
AU - Riemschneider, S
AU - Scharf, C
AU - Neubauer, P
AU - Schmid, R
AU - Hecker, M
AU - Schweder, T
PY - 2000
Y1 - 2000
N2 - The cellular response of Escherichia coli to overproduction of the insoluble heterologous protein alpha-glucosidase of Saccharomyces cerevisiae during a glucose-limited fed-batch fermentation was analyzed on the transcriptional and the translational levels. After the induction of the tac-regulated overexpression of the recombinant model protein, a significant but transient increase of the mRNA levels of the heat shock genes lon and dnaK could be observed. The mRNA level of the gene coding for the inclusion body-associated protein IbpB showed the strongest increase and remained at a clearly higher level until the end of the fermentation. By contrast, the mRNA levels of htrA and ppiB were decreased after induction of the alpha-glucosidase overexpression. Analysis of the soluble cytoplasmic protein fraction 3 h after induction revealed increased levels of the chaperones GroEL, DnaK, and Tig and a decrease in the protein levels of the two ribosomal proteins S6 and L9, the peptidylprolyl-cis-trans-isomerase PpiB, and the sigma(38)-dependent protein Dps. Analysis of the aggregated protein fraction revealed a remarkably inhomogeneous composition of the alpha-glucosidase inclusion bodies. N-terminal sequencing and MALDI-TOF mass spectrometry identification showed that most of these spots are fragments of the heterologous alpha-glucosidase. Host stress proteins, like DnaK, GroEL, IbpA, IbpB, and OmpT, have been found to be associated with the alpha-glucosidase protein aggregates.
AB - The cellular response of Escherichia coli to overproduction of the insoluble heterologous protein alpha-glucosidase of Saccharomyces cerevisiae during a glucose-limited fed-batch fermentation was analyzed on the transcriptional and the translational levels. After the induction of the tac-regulated overexpression of the recombinant model protein, a significant but transient increase of the mRNA levels of the heat shock genes lon and dnaK could be observed. The mRNA level of the gene coding for the inclusion body-associated protein IbpB showed the strongest increase and remained at a clearly higher level until the end of the fermentation. By contrast, the mRNA levels of htrA and ppiB were decreased after induction of the alpha-glucosidase overexpression. Analysis of the soluble cytoplasmic protein fraction 3 h after induction revealed increased levels of the chaperones GroEL, DnaK, and Tig and a decrease in the protein levels of the two ribosomal proteins S6 and L9, the peptidylprolyl-cis-trans-isomerase PpiB, and the sigma(38)-dependent protein Dps. Analysis of the aggregated protein fraction revealed a remarkably inhomogeneous composition of the alpha-glucosidase inclusion bodies. N-terminal sequencing and MALDI-TOF mass spectrometry identification showed that most of these spots are fragments of the heterologous alpha-glucosidase. Host stress proteins, like DnaK, GroEL, IbpA, IbpB, and OmpT, have been found to be associated with the alpha-glucosidase protein aggregates.
M3 - SCORING: Zeitschriftenaufsatz
VL - 70
SP - 217
EP - 224
JO - BIOTECHNOL BIOENG
JF - BIOTECHNOL BIOENG
SN - 0006-3592
IS - 2
M1 - 2
ER -
