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1.
Appl Microbiol Biotechnol ; 58(3): 330-7, 2002 Mar.
Article in English | MEDLINE | ID: mdl-11935184

ABSTRACT

The strong overexpression of heterologous genes in Escherichia coli often leads to inhibition of cell growth, ribosome destruction, loss of culturability, and induction of stress responses, such as a heat shock-like response. Here we demonstrate that the general stress response, which is connected to the stress response regulator sigmas (sigma38, rpoS gene product), is suppressed during strong overproduction of a heterologous alpha-glucosidase. The mRNA levels of the rpoS and osmY stress genes drastically decrease after induction of the strong overexpression system. It is shown that an rpoS mutation causes a significant loss of cell viability after induction of the expression system. Furthermore, it is demonstrated that an E. coli c/pP mutant, which could be suggested to improve heterologous protein production, is not a good production host if a tac-promoter is used to control the expression of the recombinant gene. Data from this study suggest that the overexpression of the alpha-glucosidase was greatly decreased by sigma factor competition in the clpP mutant, due to the increased sigmas level in this mutant background.


Subject(s)
Escherichia coli Proteins , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Escherichia coli/classification , Escherichia coli/metabolism , HSP70 Heat-Shock Proteins/metabolism , Recombinant Proteins/metabolism , Sigma Factor/genetics , Sigma Factor/metabolism , alpha-Glucosidases/genetics , alpha-Glucosidases/metabolism
3.
Biotechnol Bioeng ; 70(2): 217-24, 2000 Oct 20.
Article in English | MEDLINE | ID: mdl-10972933

ABSTRACT

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.


Subject(s)
Escherichia coli/metabolism , Fermentation , Glucose/metabolism , Recombinant Proteins/metabolism , Biotechnology/methods , Cell Division , Electrophoresis, Gel, Two-Dimensional , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Hydrogen-Ion Concentration , Plasmids/metabolism , RNA, Messenger/metabolism , Recombinant Proteins/genetics , Saccharomyces cerevisiae/enzymology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Time Factors , alpha-Glucosidases/metabolism
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