Monitoring of Genes that Respond to Overproduction of an Insoluble Recombinant Protein in Escherichia coli Glucose-Limited Fed-Batch Fermentations
- نوع فایل : کتاب
- زبان : انگلیسی
- مؤلف : Britta Ju¨ rgen,1 Hong Ying Lin,2 Stefan Riemschneider,2 Christian Scharf,1 Peter Neubauer,2 Roland Schmid,3 Michael Hecker,1 Thomas Schweder1
- چاپ و سال / کشور: 2000
Description
The cellular response of Escherichia coli to overproduction of the insoluble heterologous protein a-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 a-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 s38- dependent protein Dps. Analysis of the aggregated protein fraction revealed a remarkably inhomogeneous composition of the a-glucosidase inclusion bodies. Nterminal sequencing and MALDI-TOF mass spectrometry identification showed that most of these spots are fragments of the heterologous a-glucosidase. Host stress proteins, like DnaK, GroEL, IbpA, IbpB, and OmpT, have been found to be associated with the a-glucosidase protein aggregates.
BIOTECHNOLOGY AND BIOENGINEERING, VOL. 70, NO. 2, OCTOBER 20, 2000 Received 25 December 1999; accepted 27 May 2000
