Use of a design of experiments approach to optimise production of a recombinant antibody fragment in the periplasm of Escherichia coli: selection of signal peptide and optimal growth conditions

Abstract

Production of recombinant proteins such as antibody fragments in the periplasm of the bacterium Escherichia coli has a number of advantages, including the ability to form disulphide bonds, aiding correct folding, and the relative ease of release and subsequent capture and purification. In this study, we employed two N-terminal signal peptides, PelB and DsbA, to direct a recombinant scFv antibody (single-chain variable fragment), 13R4, to the periplasm via the Sec and SRP pathways respectively. A design of experiments (DoE) approach was used to optimise process conditions (temperature, inducer concentration and induction point) influencing bacterial physiology and the productivity, solubility and location of scFv. The DoE study indicated that titre and subcellular location of the scFv depend on the temperature and inducer concentration employed, and also revealed the superiority of the PelB signal peptide over the DsbA signal peptide in terms of scFv solubility and cell physiology. Baffled shake flasks were subsequently used to optimise scFv production at higher biomass concentrations. Conditions that minimised stress (low temperature) were shown to be beneficial to production of periplasmic scFv. This study highlights the importance of signal peptide selection and process optimisation for the production of scFv antibodies, and demonstrates the utility of DoE for selection of optimal process parameters.Electronic supplementary materialThe online version of this article (10.1186/s13568-018-0727-8) contains supplementary material, which is available to authorized users.

Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

“…In one study, RSM was applied to develop a defined medium to enhance human interferon gamma production (Unni et al, 2019). Using DoE, the signal peptide was selected and optimal growth conditions were established for recombinant antibody fragment production in the periplasm of E. coli (Kasli et al, 2019).…”

Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments

“…Through the literature, several attempts have been carried out to improve the production and yield of recombinant antibody fragments using these two vectors. Signal peptide sequences for these vectors have been also optimized following a stress minimization approach [ 67 , 68 ]. Karyolaimos and coworkers have obtained a yield of around 0.2 g/L of a functional scFv fragment in the periplasm using the OmpA signal peptide and 100 μM rhamnose as inductor [ 68 ].…”

“…Stress minimization results in the increased viability of cells and process robustness [ 70 , 94 ]. In the so-called Design of Experiments (DoE) setting [ 95 ] the optimization of the expression of recombinant Fab and scFv fragments in stress minimization conditions has been successfully achieved through the selection of media [ 70 , 88 , 96 , 97 ], screening of signal peptide sequences [ 67 , 68 , 75 , 98 ] and optimization of co-expressing chaperone proteins [ 56 , 88 , 99 , 100 , 101 , 102 ]. At the transcriptional level, the concept of “codon harmonization”, a sophisticated version of the codon usage optimization, has largely improved the expression of antibodies fragments [ 60 , 103 ].…”

The Rocky Road From Fed-Batch to Continuous Processing With E. coli

“…Protein secretion, commonly performed in eukaryotic cells, might also be a key solution to reducing the metabolic burden; however, recombinant protein secretion is not easy to establish in bacteria (Berlec and Strukelj, 2013; Rosano and Ceccarelli, 2014). Still, RPP in the periplasm of E. coli has shown to yield promising results, especially when producing antibody fragments, making use of the oxidizing environment (Spadiut et al, 2014a; Kasli et al, 2019). To enable protein secretion in E. coli , engineering of the Sec-Pathway is commonly performed, leading to unfolded protein secretion (Mergulhão et al, 2005; Burdette et al, 2018).…”