title: 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.
authors: Department of Bioengineering, Clemson University, 301 Rhodes Research Center, Clemson, South Carolina, 29634.
authors: Faraz
authors: F
authors: Baig
authors: Lawrence P
authors: LP
authors: Fernando
authors: Mary Alice
authors: MA
authors: Salazar
authors: Rhonda R
authors: RR
authors: Powell
authors: Terri F
authors: TF
authors: Bruce
authors: Sarah W
authors: SW
authors: Harcum
mesh_terms: D001709:Biotechnology; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000032:analysis / Q000145:classification* / Q000235:genetics* / Q000378:metabolism; D000431:Ethanol / Q000494:pharmacology; D020869:Gene Expression Profiling; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D002479:Inclusion Bodies / Q000378:metabolism*; D053858:Metabolic Networks and Pathways / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D011994:Recombinant Proteins / Q000378:metabolism*; D012995:Solubility
chemical_list: D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; D000431:Ethanol
keywords: DNA microarrays; ethanol; inclusion bodies; protein aggregates; stress