Dynamic transcriptional response of Escherichia coli to inclusion body formation

Abstract

Escherichia coli is used intensively for recombinant protein production, but one key challenge with recombinant E. coli is the tendency of recombinant proteins to misfold and aggregate into insoluble inclusion bodies (IBs). IBs contain high concentrations of inactive recombinant protein that require recovery steps to salvage a functional recombinant protein. Currently, no universally effective method exists to prevent IB formation in recombinant E. coli. In this study, DNA microarrays were used to compare the E. coli gene expression response dynamics to soluble and insoluble recombinant protein production. As expected and previously reported, the classical heat-shock genes had increased expression due to IB formation, including protein folding chaperones and proteases. Gene expression levels for protein synthesis-related and energy-synthesis pathways were also increased. Many transmembrane transporter and corresponding catabolic pathways genes had decreased expression for substrates not present in the culture medium. Additionally, putative genes represented over one-third of the genes identified to have significant expression changes due to IB formation, indicating many important cellular responses to IB formation still need to be characterized. Interestingly, cells grown in 3% ethanol had significantly reduced gene expression responses due to IB formation. Taken together, these results indicate that IB formation is complex, stimulates the heat-shock response, increases protein and energy synthesis needs, and streamlines transport and catabolic processes, while ethanol diminished all of these responses.

Global proteome response of Escherichia coli BL21 to production of human basic fibroblast growth factor in complex and defined medium

“…The metabolic burden or the cellular response toward recombinant protein production in E. coli varies greatly for different target proteins and production conditions. Many reports state enhanced levels of heat shock proteins or increased expression of heat shock genes in response to recombinant protein production . However, there are also reports that did not notice an increase of heat shock proteins or enhanced transcript levels of heat shock genes in response to protein overproduction .…”

Influence of pH control in the formation of inclusion bodies during production of recombinant sphingomyelinase-D in Escherichia coli

“…Nevertheless, when heterologous protein over-expression occurs, an inefficient folding could occur, which together with the shortage of chaperones may promote protein aggregation [2,3]. Those aggregates are called inclusion bodies [4,5], and can be formed in the cytoplasmic or periplasmic area [6,7]. IBs are dynamic reservoirs that contain a large amount of recombinant protein, various host proteins like chaperones, among other components of the cytoplasm [4,8].…”

“…Those aggregates are called inclusion bodies [4,5], and can be formed in the cytoplasmic or periplasmic area [6,7]. IBs are dynamic reservoirs that contain a large amount of recombinant protein, various host proteins like chaperones, among other components of the cytoplasm [4,8]. IBs are highly hydrated dense particles of porous structure [9,10], their surface varies from rough to smooth [6], and their size is normally in the range of 50 to 700 nm, having spherical, cylindrical or ellipsoidal teardrop shapes [10][11][12][13][14][15].…”

Awakening sleeping beauty: production of propionic acid in Escherichia coli through the sbm operon requires the activity of a methylmalonyl-CoA epimerase

“…Other studies have concluded that ScpA is similar to most mutases and can only catalyse the conversion between succinyl-CoA and the (R)-methylmalonyl-CoA enantiomer. Dayem et al [25] confirmed initial results from Vallari et al [39] that E. coli does not produce the (S)-methylmalonyl CoA enantiomer [40]. More recently, Li et al [22] engineered a succinate producing strain T110 and expressed the sbm operon and the ABb operon from Methylobacterium extorquens AM1 to produce PA.…”