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 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.
Jürgen
B
B
Institute of Microbiology, Ernst Moritz Arndt University, F.-L. Jahnstrasse 15, D-17487 Greifswald, Germany.
Lin
H Y
HY
Riemschneider
S
S
Scharf
C
C
Neubauer
P
P
Schmid
R
R
Hecker
M
M
Schweder
T
T
D001709:Biotechnology / Q000379:methods; D002455:Cell Division; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005285:Fermentation; D005947:Glucose / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D010957:Plasmids / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D000520:alpha-Glucosidases / Q000378:metabolism
D012333:RNA, Messenger; D011994:Recombinant Proteins; D000520:alpha-Glucosidases; D005947:Glucose