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.
Highlights
Recombinant protein production (RPP) is an industrially important tool for the production of hundreds of licensed recombinant proteins (RPs), including IgG antibodies and antibody fragments (Walsh 2014; SanchezGarcia et al 2016)
We show that conditions that minimise stress are favourable for production of soluble, periplasmic scFv, and demonstrate the utility of the design of experiments (DoE) approach in identifying the optimum operation window coinciding with minimum stress conditions
Previous studies have identified that: (i) lower temperatures are preferable for correct folding of RPs (Vera et al 2007), though very slow growth is observed below 20 °C; (ii) temperatures above 40 °C illicit induction of the heat shock response, Table 1 Levels for variables in the central composite design experiments
Summary
Recombinant protein production (RPP) is an industrially important tool for the production of hundreds of licensed recombinant proteins (RPs), including IgG antibodies and antibody fragments (Walsh 2014; SanchezGarcia et al 2016). Unlike their larger full-length IgG monoclonal antibody counterparts, which are commonly produced in mammalian cells, the relative simplicity of antibody fragments and their requirement for fewer post-translational modifications makes them suitable for. The periplasm of E. coli offers additional advantages as a cellular compartment for targeting RPs in bioprocesses It contains fewer proteases than the cytoplasm, which reduces the risk of proteolytic degradation during growth; and accounts for just 4–8% of the E. coli protein content (Beacham 1979). A periplasmic location affords selective extraction using approaches that disrupt or destabilise the outer membrane and cell wall, but not inner membrane (Neu and Heppel 1965; Katsui et al 1982; Naglak and Wang 1990; Weir and Bailey 1995; Kraemer et al 2016) thereby reducing demands on subsequent purification
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