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.

Keywords: DNA microarrays; ethanol; inclusion bodies; protein aggregates; stress.

Figure 1. Growth and protein production profiles for E. coli pTVP1GFP and pGFPCAT

A) Cells were cultured in minimal medium with and without induction and with and without ethanol treatment. VP1GFP (, ), GFPCAT (, ), and ethanol-treated VP1GFP (, ). Uninduced (, , ) and Induced (, , ). B) Fluorescent profiles for induced E. coli VP1GFP (, , ) and ethanol-treated induced E. coli VP1GFP (, , ) cultures. Triplicate data are shown. C) Specific CAT activity profiles for E. coli GFPCAT Uninduced () and Induced () cultures. Error bars represent 95% confidence intervals. Cultures were synchronized to Time 0 at the time of induction.

Figure 2. Protein localization withinE. colipTVP1GFP and pGFPCAT was observed using traditional and super-resolution widefield fluorescence microscopy

A) Induced E. coli pTVP1GFP (unstained); Merged DIC and fluorescence (GFP, green) images. B) Induced E. coli pGFPCAT (immunolabeled with anti-CAT antibody, shown in red). Merged DIC and fluorescence images. C) Induced E. coli pTVP1GFP (super-resolution and unstained); and D) Ethanol-treated induced E. coli pTVP1GFP (super-resolution and unstained). Areas of multiple fluorescence events in a particular location are identified in white, while fewer fluorescent events are represented in red.

Figure 3. Classical heat shock andtiggene expression profiles forE. colipTVP1GFP and pGFPCAT

The dynamic gene expression profiles for heat-shock and tig genes. VP1GFP (, ), GFPCAT (, ), and ethanol-treated VP1GFP (, ); Uninduced (, , ) and Induced (, , ). Gene expression levels were normalized to 100, which represents the “average” gene expression intensity on the DNA microarray. Standard error bars are shown.

Figure 4. Classification of genes found to be affected by protein production

These genes were identified to be significantly effected by VP1GFP and/or GFPCAT protein production by ANOVA analysis (p ≤ 0.10) followed by regression analysis (p ≤ 0.05) and/or Tukey’s W analysis (p ≤ 0.05). These genes have been grouped by function using gene ontology (GO) terms from EcoCyc and annotations from the ASAP database.

Figure 5. Gene expression profiles for protein synthesis-related genes with differential expression inE. colipTVP1GFP and pGFPCAT

A) Ribosomal subunit genes (average of 20 genes). B) Amino Acid Synthesis genes (average of 12 genes). C) Aminoacyl-tRNA Synthetase genes (average of 5 genes). The scales for panels B and C are half of the scale length used for panel A to improve visualization. VP1GFP (, ), GFPCAT (, ), and ethanol-treated VP1GFP (, ); Uninduced (, , ) and Induced (, , ). Gene expression levels were normalized to 100, which represents the “average” gene expression intensity on the DNA microarray. Standard error bars are shown.

Figure 6. Gene expression profiles for transmembrane transport and catabolism genes with differential expression inE. colipTVP1GFP and pGFPCAT

A) Transmembrane transport genes (aaeB, acrF, actP, bglF, blc, dgoT, dtpB, eamB, fucP, glpF, gltS, gntT, gspDL, lamB, malF, modB, purP, tdcC, tsgA, ugpB, uhpT, and ulaA) with decreased gene expression due to VP1GFP (average of 23 genes). B) Catabolism genes (bglB, caiB, chiA, cpdB, dgoDK, dtd, glcD, gudD, maoC, mtlD, nudE, rhaBM, tdh, tnaA, treF, uxuB, and yiaS) with decreased gene expression due to VP1GFP (average of 19 genes). VP1GFP (, ), GFPCAT (, ), and ethanol-treated VP1GFP (, ); Uninduced (, , ) and Induced (, , ). Gene expression levels were normalized to 100, which represents the “average” gene expression intensity on the DNA microarray. Standard error bars are shown.

Figure 7. Gene expression profiles for TCA cycle genes with differential expression inE. colipTVP1GFP and pGFPCAT

A) TCA cycle genes (acnB, gltA, sdhAB, and sucBC) (average of six genes). VP1GFP (, ), GFPCAT (, ), and ethanol-treated VP1GFP (, ); Uninduced (, , ) and Induced (, , ). Gene expression levels were normalized to 100, which represents the “average” gene expression intensity on the DNA microarray. Standard error bars are shown.