Abstract
ObjectivesTo reduce unwanted Fab’ leakage from an autonucleolytic Escherichia coli strain, which co-expresses OmpA-signalled Staphylococcal nuclease and Fab’ fragment in the periplasm, by substituting in Serratial nuclease and the DsbA periplasm translocation signal as alternatives.ResultsWe attempted to genetically fuse a nuclease from Serratia marcescens to the OmpA signal peptide but plasmid construction failed, possibly due to toxicity of the resultant nuclease. Combining Serratial nuclease to the DsbA signal peptide was successful. The strain co-expressing this nuclease and periplasmic Fab’ grew in complex media and exhibited nuclease activity detectable by DNAse agar plate but its growth in defined medium was retarded. Fab’ coexpression with Staphylococcal nuclease fused to the DsbA signal peptide resulted in cells exhibiting nuclease activity and growth in defined medium. In cultivation to high cell density in a 5 l bioreactor, DsbA-fused Staphylococcal nuclease co-expression coincided with reduced Fab’ leakage relative to the original autonucleolytic Fab’ strain with OmpA-fused staphylococcal nuclease.ConclusionsWe successfully rescued Fab’ leakage back to acceptable levels and established a basis for future investigation of the linkage between periplasmic nuclease expression and leakage of co-expressed periplasmic Fab’ fragment to the surrounding growth media.
Highlights
Approaching half of all global blockbuster drugs are currently antibody-based recombinant proteins, a growing number of which are Fab’ antibody fragments
A control plasmid encoding the Serratial nuclease open reading frame with no promoter upstream was readily constructed and propagated in E. coli. This suggests the Serratial nuclease coding sequence itself is not toxic to E. coli and that it is the expressed nuclease that is responsible for cytotoxicity
Consideration of the previous reports, and our own observation that Serratial nuclease fused to OmpA was lethal to cells, led us to conclude that the OmpA signal was non-functional when fused to Serratial nuclease and that this resulted in toxicity
Summary
Approaching half of all global blockbuster drugs are currently antibody-based recombinant proteins, a growing number of which are Fab’ antibody fragments. When expressed in Escherichia coli, genetically-appended peptide signals are commonly used to direct translocation of Fab’ fragment heavy and light chains to the oxidising environment of the periplasmic space where they can form disulphide bonded heterodimers (Ukkonen et al 2013). Balasundaram et al (2009) engineered E. coli so that a commercial anticancer Fab’ fragment (UCB Celltech UK, Slough, UK) was co-expressed with recombinant Staphylococcal nuclease (Fig. 1) to generate an autonucleolytic Fab’ production strain, termed ‘Fab’ Nuc’ previously and ‘OSAFab’ in this study. In the OSAFab strain, both Fab’ and nuclease were genetically fused to an OmpA periplasmic translocation peptide sequence and their transcription was controlled by Ptac promoters (de Boer et al 1983). The E. coli OmpA periplasm translocation signal directs transport to the periplasmic space via the general secretion (SEC) route (Pugsley 1993). Upon homogenisation of the OSAFab E. coli strain the Staphylococcal nuclease gained access to and degraded host DNA, decreasing the viscosity of the bioprocess stream and improving its clarification performance
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