- Original article
- Open Access
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
© The Author(s) 2019
- Received: 21 November 2018
- Accepted: 24 December 2018
- Published: 7 January 2019
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.
- Heterologous protein
- Single-chain variable fragment (scFv)
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).
The suitability of E. coli as a host for production of recombinant antibody fragments and other human biotherapeutic proteins stems, in large part from the following: (i) its physiology, metabolism and behaviour are very well understood compared to other bacterial species; (ii) it exhibits much faster growth, attains higher cell densities and also requires much cheaper growth media than mammalian hosts; and (iii) although it cannot produce RPs with ‘human-like’ glycosylation, it can generate disulphide bonds (Plückthun and Skerra 1989; Hsu et al. 2016).
Unlike the cytoplasm, which is a reducing environment, the periplasm is an oxidising environment and so favours the formation of disulphide bonds (de Marco 2009); it also contains enzymes which catalyse the formation, correction and maintenance of disulphide bonds (the Dsb enzymes (Inaba 2009). 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). Moreover, 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.
Escherichia coli possesses three major mechanisms for the transport of proteins from the cytoplasm to the periplasm. Two of these, the general secretory (Sec) and signal recognition particle (SRP) pathways, direct unfolded polypeptide chains through a protein pore in the inner membrane (the SecYEG complex), one amino acid at a time (Natale et al. 2008; Du Plessis et al. 2011; Tsirigotaki et al. 2017). In the Sec or ‘post-translational’ pathway, the polypeptide chain is translocated following complete translation by the ribosome. In the SRP or ‘co-translational’ pathway, the polypeptide chain is bound by the SRP and translocated by SecYEG whilst it is still being translated. In each case, an N-terminal signal peptide directs the polypeptide chain to the correct pathway (Freudl 2018).
During RPP processes, a key need is balancing the energy and metabolite requirements of biomass generation and RP synthesis; failure to do this has a detrimental impact on the host cell physiology, and also impairs the solubility, folding and resultant functionality of the RP (Villaverde and Carrió 2003). One common approach is to temporally segregate the biomass-production and RPP phases by employing a tightly-regulated promoter controlling RP synthesis, in combination with a chemical inducer (Overton 2014). An alternate approach is ‘stress minimisation’, whereby growth and RPP proceed concurrently, albeit at a slower rates. Practically, this is achieved by adopting lower growth temperatures and low inducer concentrations. In this way RP is translated more slowly enabling in turn more efficient folding and therefore a higher proportion of soluble, functional RP (Sevastsyanovich et al. 2009). Stress minimisation approaches have been successfully used to optimise production of cytoplasmic model proteins (Sevastsyanovich et al. 2009; Wyre and Overton 2014), cytoplasmic biotherapeutics (Selas Castiñeiras et al. 2018b) and periplasmically-targeted antibody fragments (Hsu et al. 2016; Selas Castiñeiras et al. 2018a) in fed-batch fermentation processes.
In this study a design of experiments (DoE) approach with a three-factor central composite design was used to optimise and characterise the design space for the production of a model scFv, 13R4 (Martineau et al. 1998) targeted to the periplasm of E. coli via either the SRP or SecB pathway. The use of a central composite design allowed for the minimisation of the number of cultures whilst permitting statistical analysis. The factors varied were fermentation temperature, concentration of inducer (arabinose) and the OD600 at which induction occurred; three factors known to be important in RPP (Sevastsyanovich et al. 2009; Wyre and Overton 2014; Hsu et al. 2016; Selas Castiñeiras et al. 2018b). The responses measured were the productivity, solubility and location of scFv 13R4, and measures of bacterial physiology, (Nebe-Von-Caron et al. 2000; Wyre and Overton 2014); biomass concentration (from optical density measurements); and culturability, determined using colony forming unit (CFU) counts. Models generated by this DoE approach were used to inform the design of baffled shake flask experiments, supporting higher biomass concentrations. We show that conditions that minimise stress (low temperature and inducer concentration) are favourable for production of soluble, periplasmic scFv, and demonstrate the utility of the DoE approach in identifying the optimum operation window coinciding with minimum stress conditions.
Strains and plasmids
Escherichia coli strain BL21 (F− ompT gal dcm lon hsdSB (r B − m B − ) [malB+] K-12(λS) Δ(ara)) was employed in all experiments. The plasmids used, pLBAD2-DsbA-scFv13R4+A and pLBAD2-PelB-scFv13R4+A (Selas Castiñeiras et al. 2018a), were sourced from Cobra Biologics; and encode scFv 13R4 (Martineau et al. 1998) directed to the periplasm by the E. coli DsbA and Pectobacterium carotovorum PelB signal peptides respectively, under the control of the E. coli araBAD promoter (Guzman et al. 1995). Both plasmids had the pMB1 origin of replication and encoded resistance to kanamycin.
Shake flask cultures
Overnight cultures in test tubes containing 5 mL of Luria broth (Miller, Sigma-Aldrich item L3522, UK) supplemented with 50 µg mL−1 kanamycin were inoculated with a single colony of transformed E. coli and grown for 18 h at 30 °C and 150 rpm. One millilitre of this overnight culture was used to inoculate 100 mL of Terrific Broth containing 1.2% (w/v) tryptone (Oxoid, UK), 2.4% (w/v) yeast extract (Oxoid, UK), 0.4% (v/v) glycerol, 16.9 mM KH2PO4 and 71.8 mM K2HPO4 supplemented with 50 µg mL−1 kanamycin in a 250 mL conical flask or 500 mL baffled shake flask. Shake flask cultures were grown and induced according to the conditions defined by the DoE and described in Additional file 1: Table S1, with agitation at 150 rpm. Samples of each culture were taken at time intervals for analysis. Cell pellets from [0.9/OD600] mL of culture were stored at − 20 °C until fractionation and/or SDS-PAGE.
Spectrophotometry and flow cytometry
The optical density of cultures was measured at 600 nm (OD600) using an Evolution 200 Spectrophotometer (ThermoFisher Scientific, UK). Cultures were analysed using a BD Accuri C6 flow cytometer (Becton–Dickinson, UK); bacteria were diluted in 1 mL of 0.22 µm-filtered phosphate buffered saline (PBS; Oxoid, UK) and incubated with 4 µg mL−1 propidium iodide (PI; Sigma-Aldrich, UK) and 0.1 µg mL−1 bis-(1,3-dibutylbarbituric acid) trimethineoxonol (BOX; ThermoFisher Scientific, UK) for 5 min before analysis. Data were collected at a rate of 1000–4000 events per second using a forward scatter height (FSC-H) threshold of 12,000 until 25,000 events had been recorded. PI and BOX fluorescence were measured via channels FL1-A (533/30 filter) and FL3-A (670 LP filter) respectively. Dead cell controls for PI and BOX were prepared by incubating a culture at 99 °C for 10 min prior to analysis.
Colony forming units and replica plating
Bacterial cultures were serially diluted in sterile PBS (Oxoid, UK); the two most appropriate dilutions were plated onto nutrient agar (Oxoid, UK) plates and incubated at 30 °C for 18 h before the colonies were counted. The plate containing closest to 300 colonies was stamped with sterile filter paper (Fisherbrand, USA) and stamped onto nutrient agar plates supplemented with and without 50 µg mL−1 kanamycin. After incubation at 30 °C for 18 h, colonies were counted and the percentage that had retained the plasmid was calculated.
Fractionation of soluble and insoluble proteins
To normalise the quantity of biomass for each sample, bacteria from [0.9/OD600] mL of culture were collected by centrifugation (775gav, 5 min) and resuspended in 0.25 mL of PBS (pH 8) containing 0.02% (w/v) lysozyme (ThermoFisher Scientific, UK) and 0.4% (v/v) benzonase (Sigma-Aldrich, UK). The suspensions were incubated on ice with gentle shaking for 30–45 min, then frozen in a dry ice and ethanol bath and thawed at 30 °C a minimum of three times, then centrifuged at 9000gav for 30 min. The supernatant (soluble fraction) was separated from the pellet (insoluble fraction), which was resuspended in 0.25 mL of PBS (pH 8); both fractions were stored at − 20 °C until analysis by SDS-PAGE.
Fractionation of periplasmic and spheroplast proteins
Bacteria from [0.9/OD600] mL of culture were collected by centrifugation (775gav, 5 min), resuspended in 100 µL of ice cold spheroplast buffer (100 mM Tris-HCl pH 8, 500 mM sucrose, 0.5 mM EDTA) and incubated on ice for 5 min. The cell suspensions were centrifuged at 14,000gav for 1 min, and the supernatant discarded. The pellet was resuspended by vigorous pipetting in 95 µL of ice cold 0.22 µm-filter sterilised water. After 30 s, 5 µL of 20 mM MgCl2 was added and the suspensions centrifuged at 14,000gav for 2 min. The supernatant (periplasmic fraction) was separated from pellet (spheroplast fraction). The pellet was resuspended in 100 µL of ice cold spheroplast buffer and both fractions were stored at − 20 °C until analysis by SDS-PAGE. Prior to preparation for SDS-PAGE, spheroplast fractions were incubated at 99 °C for 10 min with shaking at 500 rpm.
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and scanning densitometry
Protein compositions of fractions were analysed by reducing SDS-PAGE (Laemmli 1970) in Tris-Glycine SDS precast 12% (w/v) polyacrylamide gels (Novex WedgeWell, ThermoFisher Scientific, UK) in a Mini Gel Tank (ThermoFisher Scientific, UK) system at 200 V for 45 min. Samples containing protein were mixed with 40 µL of Tris-Glycine SDS sample buffer (63 mM Tris HCl pH 6.8, 10% (v/v) glycerol, 2% (w/v) SDS, 0.0025% (w/v) bromophenol blue), 8 μL of 500 mM DL-dithiothreitol (DTT) and 12 μL of deionised water. All samples were incubated at 85 °C for 2 min prior to loading onto the gel. Spheroplast samples were mixed with Tris-Glycine SDS sample buffer and incubated at 99 °C for 10 min before the DTT was added.
All gels were calibrated for molecular weight by loading free wells with 5 µL of an 11 to 190 kDa ladder of Blue Prestained Protein Standards (New England BioLabs, USA) of known concentration (0.2 mg/mL). After electrophoresis protein bands in gels were visualised using SimplyBlue™ Safe Stain (ThermoFisher Scientific, UK), imaged on a flatbed scanner CanoScan 9000F, Canon, UK) at a resolution of 4800 dpi, and subsequently analysed densitometrically using ImageJ software (Schneider et al. 2012) downloaded from http://rsb.infonih.gov.ij/. Productivity of scFv was determined by comparison the intensity of scFv bands to the intensity of the 25 kDa band in the 11–190 kDa reference ladder of known protein concentration. This value was then divided by the fermentation running time and calculated to give the productivity in microgram of recombinant scFv per millilitre of culture broth per hour of growth.
Design of experiments and data treatment
The design of experiment (DoE) protocol was designed in Design-Expert version 7.1 (StatEase). The design created was a circumscribed three factor, 5-level central composite design with 10 repeats; with each factor at level ‘0’, resulting in 24 runs. Once data had been collected they were checked for normality using a normal probability plot so as to ensure suitability for parametric statistical testing. The PelB productivity data were normalised by a square root transformation. The models produced were subsequently checked for significance and insignificant lack of fit. Models were used to produce contour plots, with each showing the response while two factors varied, and the third factor was maintained at level ‘0’.
RNA structure prediction
The structure of the first 100 bases of RNA encoding PelBsp-scFv and DsbAsp-scFv was predicted using the RNAfold Webserver (Lorenz et al. 2011).
Levels for variables in the central composite design experiments
OD600 at induction
[Arabinose] (% w/v)
Samples were analysed on induction and 4 h and 6 h after induction. Responses measured were: overall scFv productivity (µg scFv mL−1 culture h−1, determined by SDS-PAGE); percentage of scFv that was soluble (determined by SDS-PAGE analysis of soluble and insoluble cell fractions); percentage of scFv in the periplasmic fraction (SDS-PAGE analysis of cytoplasmic and periplasmic fractions); percentage of “healthy” cells (determined by flow cytometry and defined as having both membrane potential and membrane integrity thereby staining with neither PI nor BOX); CFU mL−1; and percentage plasmid retention (measured by replica plating). In both the DsbAsp-scFv and PelBsp-scFv systems, the responses which produced significant (p < 0.05) models with a non-significant (p > 0.05) lack of fit (tested by ANOVA) were productivity and the percentage of scFv that was soluble; both for samples taken 6 h after induction (Additional file 1: Tables S2–S5). Most other models yielded significant lack of fit.
In both systems, the fermentation temperature had a great effect on scFv productivity and also impacted on solubility, albeit to different degrees. Increasing the fermentation temperature from 20 to 40 °C was met with improved whole cell productivity in the case of DsbAsp-scFv, whereas optimal productivity was observed at ca. 32 °C for the PelBsp-scFv system. The observed enhancement in productivity at higher temperatures, likely due to faster rates of protein translocation and transcription, and faster growth leading to earlier induction, come at the expense of reduced scFv solubility.
In summary, DsbAsp generated high scFv productivity, but the solubility and periplasmic targeting of scFv, and overall cell physiology (membrane potential, membrane integrity and culturability) were poor. Conversely scFv productivity from PelBsp was much lower cf. DsbAsp, but scFv solubility and periplasmic targeting were all high; cell physiology was also better than for DsbAsp. For these reasons, PelBsp was chosen as the signal peptide for further optimisation.
Intensification to baffled shake flasks
Lower growth temperature and late induction at high biomass favoured high-level scFv production, high periplasmic concentrations (Fig. 7d) and preferential accumulation of scFv in the periplasm (Fig. 7e). The concentration of periplasmic scFv (Fig. 7d) reached maximal levels of 20 μg mL−1 at 25 °C 6 h after induction at high biomass concentration, and nearly 80% of the total scFv was periplasmic (Fig. 7e). The overall concentration of periplasmic scFv generated here is comparable to concentrations of Fab fragment D1.3 under stress-minimised conditions (Hsu et al. 2016); production of periplasmic Fab was likewise highest at low growth temperature.
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.
For both DsbAsp and PelBsp, temperature was the main influence on scFv concentration and solubility, with higher temperatures giving rise to increased scFv productivity, but lower scFv solubility, likely caused by an imbalance between the rates of scFv translation and translocation. Previous studies have shown that the SecYEG translocon is a bottleneck for periplasmic RPP (Schlegel et al. 2013). If the SecYEG pore becomes overloaded, both RP and native proteins are prevented from passing into the periplasm, leading to misfolding in the cytoplasm and deleterious effects on bacterial physiology. Therefore, balancing the rate of RP translation with that of translocation to the periplasm is critical. Similar results were previously observed with a Fab antibody fragment (Hsu et al. 2016).
Selection of signal peptide is known to be in important step for design of processes where RP is targeted to the periplasm (Freudl 2018); currently, in the absence of ways to accurately predict the best signal peptide to use, selection must be determined experimentally (Selas Castiñeiras et al. 2018a). In this study, whereas the DsbA signal peptide, targeting scFv to the periplasm via the co-translational SRP route, generated higher concentrations of scFv, this was at the expense of scFv solubility and bacterial physiology (membrane potential, membrane integrity and culturability). Therefore the PelB signal peptide (directing scFv to the periplasm via the post-translational SecB pathway), which gave rise to lower scFv concentrations, but far higher scFv solubility and better cell physiology, was the preferred signal peptide and was chosen as the signal peptide for further optimisation.
Growth at higher biomass concentration in baffled shake flasks at 25 and 35.5 °C revealed that optimal periplasmic scFv production occurred at the lower temperature and induction at a high biomass concentration (OD600 = 25). We envisage this information will prove useful in future work to develop bioreactor-based high cell density fermentations for scFv production. Overall, these findings confirm previous studies on stress minimisation for RPP, both for cytoplasmic (Sevastsyanovich et al. 2009; Wyre and Overton 2014; Selas Castiñeiras et al. 2018b) and periplasmic targeting (Hsu et al. 2016; Selas Castiñeiras et al. 2018a).
IMK performed the practical work and analysed the data, supported by TWO and ORTT. IMK, ORTT and TWO designed the programme of work and wrote the manuscript. All authors read and approved the final manuscript.
The authors would like to thank Tania Selas Castiñeiras, Steve Williams, Tony Hitchcock and Daniel Smith from Cobra Biologics for materials and their input into the project.
The authors declare that they have no competing interests.
Availability of data and materials
Data is available within this article and the accompanying supplemental materials. Data and materials can also be requested from the corresponding author.
Consent for publication
Ethics approval and consent to participate
This study did not involve human participants or animals.
This study was funded by a UK Biotechnology and Biological Sciences Research Council Ph.D studentship to IMK as part of the Midlands Integrative Bioscience Training Partnership scheme. The funders played no role in the design of the study, the collection, analysis or interpretation of the data, or the writing of the manuscript.
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