title: The transcriptional response of Escherichia coli to recombinant protein insolubility.
abstract: Bacterial production of recombinant proteins offers several advantages over alternative expression methods and remains the system of choice for many structural genomics projects. However, a large percentage of targets accumulate as insoluble inclusion bodies rather than soluble protein, creating a significant bottleneck in the protein production pipeline. Numerous strategies have been reported that can improve in vivo protein solubility, but most do not scale easily for high-throughput expression screening. To understand better the host cell response to the accumulation of insoluble protein, we determined genome-wide changes in bacterial gene expression upon induction of either soluble or insoluble target proteins. By comparing transcriptional profiles for multiple examples from the soluble or insoluble class, we identified a pattern of gene expression that correlates strongly with protein solubility. Direct targets of the sigma32 heat shock sigma factor, which includes genes involved in protein folding and degradation, were highly expressed in response to induction of insoluble protein. This same group of genes was also upregulated by insoluble protein accumulation under a different growth regime, indicating that sigma32-mediated gene expression is a general response to protein insolubility. This knowledge provides a starting point for the rational design of growth parameters and host strains with improved protein solubility characteristics. Summary Problems with protein solubility are frequently encountered when recombinant proteins are expressed in E. coli. The bacterial host responds to this problem by increasing expression of the protein folding machinery via the heat shock sigma factor sigma32. Manipulation of the sigma32 regulon might provide a general mechanism for improving recombinant protein solubility.
authors.affiliation: Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville, MD 20850, USA smithh@umbi.umd.edu
authors.forename: Harold E
authors.initials: HE
authors.lastname: Smith
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry; D001671:Biochemistry / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D015964:Gene Expression Regulation, Bacterial; D006360:Heat-Shock Proteins; D018869:Heat-Shock Response; D020411:Oligonucleotide Array Sequence Analysis; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry*; D011995:Recombination, Genetic; D012808:Sigma Factor / Q000737:chemistry*; D012995:Solubility; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; D012808:Sigma Factor; C069774:heat-shock sigma factor 32