Improving Fab’ fragment retention in an autonucleolytic Escherichia coli strain by swapping periplasmic nuclease translocation signal from OmpA to DsbA

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.

Harnessing the potential of bacterial oxidative folding to aid protein production

“…The DsbA SS outperforms other signal peptides for the recombinant expression of several proteins, such as Fbs1 (Chen & Samuelson, 2016), roGH (Durrani et al., 2015) and hGH (Soares et al., 2003), mPRL (Suzuki et al., 2012), as well as potato and tick carboxypeptidase inhibitors (Puertas et al., 2011). Moreover, it has been used for the secretion of engineered single‐domain antibodies, such as scFvs, VNARs, and VHHs, with mixed success (Fink et al., 2019; Kasli et al., 2019; Leow et al., 2019; Monegal et al., 2012; Schofield et al., 2017; Thie et al., 2008). It is unclear what determines the suitability of the DsbA SS for each application, but the positive charge of the n‐region and the hydrophobicity of the h‐region (Figure 2, top), both important factors for successful protein translocation, have been modified for this signal peptide to improve export efficiency (Han et al., 2017; Zhou et al., 2016).…”

Scale-Up and Bioprocessing of Phages

“…When the cells are lysed by a bacteriophage such as λ or by homogenisation, the nuclease can gain access to the released DNA and RNA and degrade them. This 'cell engineering' approach to assisting bioprocessing was developed at UCL and has been shown to give considerable gains in the centrifugation steps and other downstream purification steps in bioprocessing of proteins such as Fab fragments [33][34][35].…”