Abstract
BackgroundThe overexpression of scFv antibody fragments in the periplasmic space of Escherichia coli frequently results in extensive protein misfolding and loss of cell viability. Although protein folding factors such as Skp and FkpA are often exploited to restore the solubility and functionality of recombinant protein products, their exact impact on cellular metabolism during periplasmic antibody fragment expression is not clearly understood. In this study, we expressed the scFvD1.3 antibody fragment in E. coli BL21 and evaluated the overall physiological and global gene expression changes upon Skp or FkpA co-expression.ResultsThe periplasmic expression of scFvD1.3 led to a rapid accumulation of insoluble scFvD1.3 proteins and a decrease in cell viability. The co-expression of Skp and FkpA improved scFvD1.3 solubility and cell viability in a dosage-dependent manner. Through mutagenesis experiments, it was found that only the chaperone activity of FkpA, not the peptidyl-prolyl isomerase (PPIase) activity, is required for the improvement in cell viability. Global gene expression analysis of the scFvD1.3 cells over the chaperone-expressing cells showed a clear up-regulation of genes involved in heat-shock and misfolded protein stress responses. These included genes of the major HSP70 DnaK chaperone family and key proteases belonging to the Clp and Lon protease systems. Other metabolic gene expression trends include: (1) the differential regulation of several energy metabolic genes, (2) down-regulation of the central metabolic TCA cycle and transport genes, and (3) up-regulation of ribosomal genes.ConclusionsThe simultaneous activation of multiple stress related and other metabolic genes may constitute the stress response to protein misfolding in the scFvD1.3 cells. These gene expression information could prove to be valuable for the selection and construction of reporter contructs to monitor the misfolded protein stress response during antibody fragment production.
Highlights
The overexpression of scFv antibody fragments in the periplasmic space of Escherichia coli frequently results in extensive protein misfolding and loss of cell viability
E. coli BL21 cells carrying the pET-scFvD1.3 or a blank pET plasmid were cultured in 2 L bioreactors to late-log phase before isopropyl β-D-1-thiogalactopyranoside (IPTG) induction of scFvD1.3 expression
The periplasmic expression of scFvD1.3 antibody fragments led to the accumulation of insoluble scFvD1.3 proteins and a decrease in the cell viability of scFvD1.3 cells, providing a model to examine the metabolic stress possibly related to protein misfolding and its associated loss of cell viability
Summary
The overexpression of scFv antibody fragments in the periplasmic space of Escherichia coli frequently results in extensive protein misfolding and loss of cell viability. Protein folding factors such as Skp and FkpA are often exploited to restore the solubility and functionality of recombinant protein products, their exact impact on cellular metabolism during periplasmic antibody fragment expression is not clearly understood. The periplasmic localization of several protein folding factors and chaperones catalyze the proper assembly and folding of functional scFv antibody fragments [7,8]. The co-expression of FkpA with scFv significantly improved the latter's soluble and functional expression [16] These protein folding factors are increasingly exploited to improve the soluble expression of recombinant protein products in the periplasm, the detailed impact on host cell metabolism is still not clearly understood
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