Monitoring of genes that respond to overproduction of an insoluble recombinant protein in Escherichia coli glucose-limited fed-batch fermentations

The cellular response of Escherichia coil to overproduction of the insolubl e heterologous protein oc-glucosidase of Saccharomyces cerevisiae during a glucose-limited fed-batch fermentation was analyzed an the transcriptional and the translational levels. After the induction of the tac-regulated over expression of the recombinant model protein, a significant but transient in crease of the mRNA levels of the heat shock genes ion and dnaK could be obs erved. 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 h trA and ppiB were decreased after induction of the alpha-glucosidase overex pression. Analysis of the soluble cytoplasmic protein fraction 3 h after in duction revealed increased levels of the chaperones GroEL, DnaK, and Tig an d 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 remarka bly inhomogeneous composition of the alpha-glucosidase inclusion bodies. N- terminal sequencing and MALDI-TOF mass spectrometry identification showed t hat most of these spots are fragments of the heterologous alpha-glucosidase . Host stress proteins, like DnaK, GroEL, IbpA, IbpB, and OmpT, have been f ound to be associated with the alpha-glucosidase protein aggregates. (C) 20 00 John Wiley & Sons, Inc.