Co-expression of Skp and FkpA chaperones improves cell viability and alters the global expression of stress response genes during scFvD1.3 production

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.

Engineering toward a bacterial “endoplasmic reticulum” for the rapid expression of immunoglobulin proteins

“…FkpA has been reported to improve protein solubility as well as catalyze prolyl isomerization for several model proteins. 14,[19][20][21] In addition, FkpA localized to the cytoplasm has also been shown to enhance secretion of both Fabs and scFv into the E. coli periplasm, possibly by helping each stay soluble long enough for export from the cytoplasm. 22 However, FkpA alone is insufficient to support IgG assembly (Fig.…”

Engineering production of functional scFv antibody in E. coli by co-expressing the molecule chaperone Skp

“…It was also reported that the solubility and affinity of scFv was improved by co-expression of molecular chaperones such as Skp, Dsbc, and FkpA (Ow et al, 2010; Sonoda et al, 2011). In some cases, co-expression of molecular chaperone not only improves the soluble expression but also increases the cell viability (Ow et al, 2010).…”

Role for Skp in LptD Assembly in Escherichia coli

“…FkpA has long been known to act as a chaperone for nonnative or mutant E. coli proteins (29)(30)(31), but until recently, its physiological role was largely unclear. It has been shown that FkpA is necessary for colicin M toxicity in vivo (32,33) and that its PPIase activity is required for this function (34).…”