Novel Cell Response to Inclusion Body Formation in Escherichia coli

Recombinant proteins are overexpressed in Escherichia coli to produce large amounts of proteins for therapeutic drugs, industrial enzymes, and other applications. Often these valuable proteins become malformed, less active aggregates referred to as inclusion bodies (IBs). Although many studies have focused on preventing inclusion body formation, there are few studies that examine the cellular response to IBs.  We hypothesize that the cell response will reveal the mechanisms that precede recombinant protein aggregation. Current bioanalytical techniques do not provide an adequate picture of inclusion body formation and the cell response. We compared two similar GFP-fusion proteins with different solubility by using a combination of fluorescence, protein, nucleic acid, and transcription analysis to assess the cell response. Fluorescent microscopy images of E. coli with aggregate protein had distinctive cytoplasmic features that verified the presence IBs. DNA microarrays identified the transcription response during the IB formation and also in the presence of chemical additions. We identified novel genes that participate in the E. coli stress response to IBs and chemical treatment. In contrast to cells expressing soluble recombinant proteins, the cells with IBs had significant upregulation in genes encoding uncharacterized proteins.  The overall benefit from this study will lead to biological or chemical approaches to improve recombinant protein production and lower aggregation.