Date Published: January 7, 2019
Publisher: Springer Berlin Heidelberg
Author(s): Ikhlaas M. Kasli, Owen R. T. Thomas, Tim W. Overton.
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.
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; Sanchez-Garcia 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 production in bacterial hosts. The bacterium Escherichia coli is a commonly employed host for recombinant protein production (RPP) contributing to the production of one-third of FDA approved human biotherapeutics (Overton 2014; Walsh 2014). Single chain variable fragments (scFv) are an emerging class of IgG fragments comprising the antigen-binding variable heavy (VH) and variable light (VL) domains fused into a single polypeptide chain with a flexible linker (Nelson 2010).
A DoE approach using a central composite design was employed to optimise the production of the model scFv 13R4 (Martineau et al. 1998), henceforth referred to as scFv. Two plasmids were used to target scFv to the periplasm, one using the E. coli DsbA signal peptide, DsbAsp (Schierle et al. 2003) and the SRP pathway, the other employing Pectobacterium carotovorum PelB signal peptide, PelBsp (Lei et al. 1987) and the SecB pathway. The arabinose-inducible pBAD promoter (Guzman et al. 1995) regulated scFv expression from both plasmids. The variables in these experiments were: temperature (20.6 °C to 39.9 °C); the concentration of arabinose used to induce recombinant protein production (0 to 0.26% w/v, the pBAD user manual suggests a maximum arabinose concentration of 0.2% (Invitrogen 2010); and the OD600 at which induction occurred, between 0.29 (early exponential) and 1.21 (early stationary phase). Levels for each variable are shown in Table 1. Preliminary experiments confirmed that these ranges were suitable for these strains and growth 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, potentially increasing proteolysis (Meyer and Baker 2011); and (iii) a temperature of 25 °C is a good compromise between growth and RP folding (Sevastsyanovich et al. 2009). All cultures were grown in 100 mL of terrific broth with 0.4% (w/v) glycerol as a carbon source in 250 mL conical flasks. Additional file 1: Table S1 lists the growth conditions of each individual culture.Table 1Levels for variables in the central composite design experimentsVariableLevel− α− 10+ 1+ αTemperature (°C)30.625.030.335.539.9OD600 at induction0.290.500.751.001.21[Arabinose] (% w/v)0.0000.0590.1300.2000.259
The design of experiments (DoE) approach employed in this study to optimise process conditions influencing bacterial physiology and the productivity, solubility and location of scFv: (i) highlighted that titre and subcellular location of the scFv depend on the temperature and inducer concentration employed; and (ii) revealed the superiority of the PelB over the DsbA signal peptide in terms of scFv solubility and cell physiology.