Teaching an old pET new tricks: tuning of inclusion body formation and properties by a mixed feed system in E. coli

Abstract

Against the outdated belief that inclusion bodies (IBs) in Escherichia coli are only inactive aggregates of misfolded protein, and thus should be avoided during recombinant protein production, numerous biopharmaceutically important proteins are currently produced as IBs. To obtain correctly folded, soluble product, IBs have to be processed, namely, harvested, solubilized, and refolded. Several years ago, it was discovered that, depending on cultivation conditions and protein properties, IBs contain partially correctly folded protein structures, which makes IB processing more efficient. Here, we present a method of tailored induction of recombinant protein production in E. coli by a mixed feed system using glucose and lactose and its impact on IB formation. Our method allows tuning of IB amount, IB size, size distribution, and purity, which does not only facilitate IB processing, but is also crucial for potential direct applications of IBs as nanomaterials and biomaterials in regenerative medicine.

Keywords: Escherichia coli BL21(DE3); Inclusion body properties; Inclusion body purity; Inclusion body size; Lactose; pET expression system.

Fig. 1

Tuning IB formation rate. a Black line indicates the maximum specific uptake rate of lactose (q _s,lac) as a function of the specific uptake rate of glucose (q _s,glu) for an Escherichia coli BL21(DE3) strain producing enhanced green fluorescent protein (eGFP). Data points (open circles) were obtained from several batch and fed-batch cultivations and fitted by the mechanistic model according to our previous study (Wurm et al. 2016, 2017). Colored symbols indicate performed experiments as shown in (b) and (c). Error bars indicate deviation of the respective q _s over induction time. b Specific IB titer in mg_eGFP/g_cells as a function of time for lactose and IPTG (0.5 mM) induction. c Summary of specific sugar uptake rates (q _s) and specific IB formation rates (q _p,IB). The error bars of the specific IB titers indicate the standard deviation (namely 11.25%), which was identified by performing biological replicates of the center point (i.e., 18% q _s,lac,max)

Fig. 2

Tuning the size of IBs. a Scanning electron microscopy pictures of IBs from different cultivations used to asses IB size, as exemplarily shown in lower right figure. Percentage indicates proportion of maximum specific lactose uptake rate (q _s,lac) used for induction. Red scale bars: 5 μm. 3.5-fold zoom for IPTG induction (lower right). b (i) exemplary atomic force microscopy picture of typical IBs showing spherical shape, (ii) and (iii) zoom in on IB particle, and (iv) topography cross-section of an isolated IB (indicated as a blue line in iii). c Probability density plot of IB size distribution after 12 h of induction as a function of q _s,lac showing that IB size can be tuned by q _s,lac. Red-dashed line indicates logarithmic fit between IB size and q _s,lac (degree of freedom = 2, R ² = 0.99). IB diameter with standard deviation from different cultivations after 12 h of induction are shown in the table. Standard deviation was evaluated from measuring 50 IBs per sample

Fig. 3

Size distribution of IBs over induction time. a Probability density plot of IB size distribution as a function of induction time indicating that IB size increases, while also, the distribution gets broader over time for different induction conditions. b IB diameter with standard deviation from different cultivations conditions at different time points of induction

Fig. 4

Secondary structure of IBs measured by infrared (IR) spectroscopy. a IR spectra of IBs from different induction regimes. Maxima for β-sheet secondary structure appear at approx. 1630 and 1690 cm⁻¹ in the IR spectrum, whereas the shoulder at approx. 1655 cm⁻¹ is attributed to α-helical secondary structure. b Table shows degree of spectral overlap (s _1,2) for IBs from different induction regimes (4% q _s,lac,max (small IBs, Ø = 408 nm); IPTG (medium IBs, Ø = 490 nm); and 57% q _s,lac,max (large IBs, Ø = 600 nm)) calculated according to Schwaighofer et al. (Schwaighofer et al. 2016) demonstrating a very high degree of spectral overlap for all samples. The value of s _1,2 ranges from 0 to 1, corresponding to no overlapping and complete overlapping, respectively

Fig. 5

Impact of IB size on IB purity. a Purity determined by HPLC impurity monitoring using size exclusion chromatography (SEC) after solubilization with 2 M urea of IBs with a small (Ø = 408 nm), medium (Ø = 490 nm) and large (Ø = 600 nm) diameter. Standard deviation was evaluated from technical duplicates. b Purity of eGFP determined by HPLC impurity monitoring using SEC after refolding. Standard deviation was evaluated from technical duplicates. c Overview of results from solubilization and refolding with standard deviations