Downstream processing of Cry4AaCter-induced inclusion bodies containing insect-derived antimicrobial peptides produced in Escherichia coli

Protein Expression and Purification 155 (2019) 120–129

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Downstream processing of Cry4AaCter-induced inclusion bodies containing insect-derived antimicrobial peptides produced in Escherichia coli

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Daniel Hoffmanna, Dustin Eckhardta, Doreen Gerlachb, Andreas Vilcinskasb,c, Peter Czermaka,b,c,d,∗ a

University of Applied Sciences Mittelhessen, Institute of Bioprocess Engineering and Pharmaceutical Technology, Wiesenstrasse 14, 35390, Giessen, Germany Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Project Group Bioresources, Heinrich-Buff-Ring 26, 35392, Giessen, Germany c Justus Liebig University, Heinrich-Buff-Ring, 35392, Giessen, Germany d Kansas State University, Faculty of Chemical Engineering, 1005 Durland Hall 1701A Platt Street, Manhattan, KS, 66506, USA b

ARTICLE INFO

ABSTRACT

Keywords: Resolubilization Crossflow filtration Insect metalloprotease inhibitor Pull-down tag Endotoxin removal Polyether sulfone membranes

The Cry4AaCter tag is a pull-down tag which promotes the formation of inclusion bodies (IBs) that can be resolubilized in an alkaline buffer. Here, we used the Cry4AaCter tag to create a platform for the production of antimicrobial peptides (AMPs) in Escherichia coli featuring a uniform resolubilization process independent of the peptide fused to the pull-down tag. The Cry4AaCter tag conserves the bioactivity of fusion proteins and thus allows the purification of simple AMPs and more complex AMPs stabilized by disulfide bonds. We developed a downstream process (DSP) for the purification of IBs containing the mutated Galleria mellonella insect metalloprotease inhibitor IMPI(I38V), which has a globular structure stabilized by five disulfide bonds. IMPI(I38V) is a potent inhibitor of the M4 metalloproteases used as virulence factors by several human pathogens. We used a single crossflow filtration for the washing and resolubilization of the Cry4AaCter-induced IBs and obtained bioactive IMPI(I38V) after tag removal. We achieved a 68-fold higher protein yield using our IB system compared to an alternative DSP approach in which a GST-fusion strategy was used to produce soluble IMPI(I38V). The Cry4AaCter-based process was transferable to gloverin (another G. mellonella AMP) and the visible marker green fluorescent protein, which accumulated in fluorescent IBs, confirming it is a broadly applicable strategy for the recovery of functional proteins.

1. Introduction The increasing prevalence of antibiotic-resistant pathogens is a pressing healthcare challenge [1,2]. Antimicrobial peptides (AMPs) have been proposed as one potential solution because although microbes can evolve resistance against AMPs [3], it is possible to use combinations of AMPs [4,5], AMPs together with conventional antibiotics [6,7], and AMP fusions with other protective proteins and peptides to reduce the likelihood of resistance [8–10]. Insects possess the most diverse spectrum of AMPs and offer a valuable resource for the isolation of peptides that can tackle multidrug-resistant microbes and even some types of cancer [11–13]. Simple AMPs can be produced by solid-phase chemical synthesis [7]. However, this is not feasible for

more complex AMPs and the most suitable approach is the production of recombinant peptides in microbes, or insect cell lines given that these represent the native production host [14]. Microbes such as Escherichia coli are suitable for AMP production if the function of the peptide does not rely on glycosylation or the formation of disulfide bonds, although the latter can be accommodated by E. coli mutant strains with an oxidizing cytoplasmic environment [15]. Many AMPs are toxic towards E. coli, but only in their soluble form. Therefore, high yields can be achieved without toxicity if the AMP is produced as insoluble inclusion bodies (IBs). An efficient isolation method for IBs can be found by high-throughput screening [16]. However, the main problem with IBs is the efficient recovery of the soluble, active form of the protein. The classical approach begins with

Abbreviations: AMP, antimicrobial peptide; DSP, downstream processing; EDTA, ethylenediaminetetraacetic acid; GFP, green fluorescent protein; GST, glutathioneS-transferase; IB, inclusion body; IMPI, Insect metalloprotease inhibitor; IPTG, isopropyl-β-D-thiogalactopyranoside; LPS, lipopolysaccharide; PBS, phosphate buffered saline; PES/PESU, polyethersulfone; rfu, relative fluorescence units; TBS, Tris-buffered saline; TCA, trichloroacetic acid; TMP, transmembrane pressure ∗ Corresponding author. University of Applied Sciences Mittelhessen, Institute of Bioprocess Engineering and Pharmaceutical Technology, Wiesenstrasse 14, 35390, Giessen, Germany. E-mail address: [email protected] (P. Czermak). https://doi.org/10.1016/j.pep.2018.12.002 Received 9 August 2018; Received in revised form 29 November 2018; Accepted 3 December 2018 Available online 04 December 2018 1046-5928/ © 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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the denaturation of IBs using high concentrations of chaotropic reagents such as urea and guanidine hydrochloride, followed by the removal of the chaotrope by dialysis in a buffer that promotes refolding. The refolding phase often takes many hours and the composition of the buffer needs to be determined empirically for each protein. This classical approach is therefore cumbersome and expensive, and the yield losses can be high [17]. Challenges associated with classical IBs can be addressed by using pull-down fusion tags that promote IB formation, because this enables new DSP strategies [18,19]. For example, the Cry4AaCter pull-down tag enables the pH-dependent resolubilization of IBs containing the fusion protein [20]. The 18 kDa Cry4AaCter tag can be fused to recombinant proteins as either an N-terminal [20–22] or C-terminal [20,23] tag, and in all but one case it achieved the formation of IBs that were amenable to pH dependent resolubilization [21]. The bioactivity of glutathione-Stransferase (GST) was detected after resolubilization without a refolding step, and various insecticidal proteins were also active after resolubilization [20,22,23]. The simple pH-dependent resolubilization of these deliberately generated IBs and the size of IBs [24] suggest that membrane-based purification may be feasible. IBs have already been washed and resolubilized in a crossflow filtration unit using the classical denaturation approach, making the dilution of the chaotropes a less expensive step [25]. Here, we established a downstream processing (DSP) strategy for Cry4AaCter-based IBs using a single crossflow filtration unit for washing and resolubilization. A DSP platform combining the unified resolubilization method of the Cry4AaCter tag with a simple filtration to separate a large amount of host cell protein from the fusion protein of interest would be very useful as first purification step for various AMP purifications and could also be used for more complex proteins of interest. The fusion protein of interest was a mutated version of the insect metalloprotease inhibitor known as IMPI(I38V). IMPI was first isolated from larvae of the greater wax moth (Galleria mellonella) challenged with chemical mimetics of bacterial or fungal pathogens [26]. It was identified as a potent and selective competitive inhibitor of M4 metalloproteases [27], which are virulence factors in many pathogens [28]. IMPI(I38V) has a molecular mass of 7.8 kDa and a globular structure stabilized by five disulfide bonds, which enables its repeated recognition as a M4 metalloprotease substrate [27]. The mutation IMPI (I38V) was designed in silico and is one of several mutations of IMPI, expected to show increased inhibitory activity. We used this mutation as a model peptide for the proof of principle of the new DSP approach. We produced IMPI(I38V) as insoluble IBs using the Cry4AaCter tag and also as a soluble fusion protein using the GST tag for solubility enhancement. Soluble production of GST-IMPI(I38V) in E. coli was possible because the antimicrobial effect of IMPI is not direct but indirect by inhibiting secreted proteases. We recovered the recombinant protein to compare the yields of both processes. We also investigated the transferability of the Cry4AaCter system by using it to produce another G. mellonella AMP (gloverin) and the visual marker green fluorescent protein (GFP).

2.2. Production of recombinant fusion proteins The production strains (Merck, Darmstadt, Germany) were all DE3 lysogens that expressed the T7 RNA polymerase gene upon induction with isopropyl-β-D-galactopyranoside (IPTG) (Sigma-Aldrich). The E. coli Rosetta-gami 2(DE3) pLysS strain was used because it can form disulfide bonds in the cytoplasm, whereas BL21(DE3) was used because it has a reducing cytoplasm. Cultures were grown at 37 °C in 1-L baffled shaker flasks containing 100 ml terrific broth medium (Carl Roth, Karlsruhe, Germany) supplemented with 5 g l−1 glycerol (Carl Roth) shaking at 250 rpm in a Multitron shaker (Infors, Bottmingen, Switzerland), with an initial OD600 of ∼0.1. The cultures were induced with 1 mM IPTG when the OD600 reached ∼1.0. The cells were harvested 4 h later by centrifugation (15294 x g, 10 min, 4 °C) and pellets were stored at −20 °C. Bioreactor cultivations were carried out using a double-walled glass vessel with a 5-L working volume connected to an ez-Control unit (Applikon, Delft, Netherlands). The temperature was set to 37 °C and controlled with a heating sleeve and water cooling. The pH was set to 7.0 and controlled with 1 M NaOH. The presence of foam was detected with a conductive probe and the foam level was controlled by the addition of Antifoam C Emulsion (Sigma-Aldrich). The aeration rate was set to 1 vvm with air and three Rushton stirrers were used, initially at 400 rpm. The lower limit of dissolved oxygen was set to 55% and a stirrer ramp up to 850 rpm controlled the level of dissolved oxygen. As above, cultures with an initial OD600 of ∼0.1 were induced when the OD600 reached ∼1.0 and were harvested by centrifugation 4 h later (17207 x g, 10 min, 4 °C) and the pellets stored at −20 °C. 2.3. Cell lysis and IB isolation Cell pellets from shake-flask cultivations were chemically lysed with BugBuster Master Mix (Merck) according to the manufacturer's instructions and IB fractions were isolated according to the manufacturer's protocol and stored at −80 °C. Cell pellets from bioreactor cultivations were resuspended at 100 g wet weight L−1 in buffer A (50 mM Tris-HCl, 150 mM NaCl, pH 7.0) and mechanically lysed by high pressure homogenization (EmulsiFlex-C5, Avestin, Ottawa,Canada) with two passages at 100–130 MPa. The flow-through was placed on ice. The soluble and insoluble fractions were separated by centrifugation (17207 x g, 30 min, 4 °C) and if IBs were present the insoluble fraction was stored at −20 °C. For the production of soluble GST-IMPI(I38V), the supernatant was processed immediately. 2.4. Sodium tetraborate precipitation For proof-of-principle experiments, the 750 μl IB fraction was mixed 1:1 with 0.1 M sodium tetraborate (Merck) in 2-ml reaction tubes and incubated on a rotary shaker for 1 h at room temperature. The samples were centrifuged (1000 x g, 2 min, 22 °C) and 90% of the supernatant was discarded. The pellet was resuspended in the remaining 10% of buffer and transferred to a new reaction tube. The volume was made up to 1.5 ml with buffer A. Precipitation in 50-ml tubes was carried out in a similar manner but the centrifugation time was prolonged. Due to the greater sedimentation distance, a factor of 3.643 was considered. An increase of the centrifugation radius from 0.055 m to 0.095 m resulted in an increase in the necessary sedimentation time according to Equation (1), where V is the effective particle velocity, which was assumed to stay constant, r is the centrifugation radius, and t is the centrifugation time. Based on the empirical centrifugation time of 120 s with the smaller radius, the equation was used to calculate the new centrifugation time with the larger radius, resulting in a factor of 1.727.

2. Materials and methods 2.1. Expression vectors Expression vectors were cloned using the modular Golden Gate system [29]. All vectors contained the T7 promoter and terminator and a gentamycin resistance marker. Positive clones were selected on 15 μg ml−1 gentamycin sulfate (Sigma-Aldrich, St. Louis, USA). Each vector also contained the coding sequence for a thrombin cleavage site between the fusion protein and the protein of interest.

V=

2

r t

1

Both factors were combined to calculate the centrifugation time of 12 min 30 s for the precipitation in 50-ml tubes. 121

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2.5. Screening of wash buffers

was exchanged for TBS in 5-ml gel filtration columns as described above.

Wash buffers for lipopolysaccharide (LPS) reduction were evaluated using 1.5-ml aliquots of BL21(DE3) lysate after the production of Cry4AaCter-IMPI(I38V) IBs. The samples were centrifuged (1000 x g, 20 min, 22 °C) and the pellets were resuspended in water. Samples of the lysate and the IB fraction in water were taken for LPS measurements. The IB fraction was then washed twice by centrifugation (1000 x g, 20 min, 22 °C) using alternative wash buffers for LPS removal: 1 mM ethylendiaminetetraacetic acid (EDTA) [30], 0.5% (v/v) Triton X-100 [31,32], 0.5% (v/v) Tween-20 [32], and 0.3 M arginine [30]. The pellet was finally resuspended in water. Two washes with water were carried out as a control.

2.9. Analytics 2.9.1. Measurement of protein concentration Protein concentrations in polyacrylamide gels were determined by densitometry, with bovine serum albumin as a standard. Band intensities were compared using ImageLab v5.2.1 (Bio-Rad). Purified IMPI(I38V) was quantified by photometry using a Synergy HTX plate reader and a take-3 microspot plate (BioTek Instruments, Winooski, USA) with a extinction coefficient of 6585 M−1 cm−1 determined using ProtParam.

2.6. IB resolubilization and tag removal

2.9.2. SDS-PAGE Samples were loaded onto stain-free 4–20% TGX SDS gradient gels (Bio-Rad) and SDS-PAGE was carried out using a Criterion Cell with a Power Pack Basic (Bio-Rad) at 120 V for 45 min with Tris-glycine-SDS buffer (Bio-Rad). We used 5 μl Precision Plus Protein unstained marker (Bio-Rad) as molecular weight standards. Protein bands were visualized using the ChemiDoc MP imaging system (Bio-Rad) with UV activation for 5 min.

Resolubilization was carried out as previously described [20]. IB fractions were thawed at room temperature and resuspended in TBS using 5 ml g−1 wet weight of the cell pellet from which the IBs were isolated. The suspension was centrifuged (15294 x g, 10 min, 22 °C) and resuspended in the same volume of 50 mM NaHCO3 (pH 11.0) while rotating in the Multitron shaker at 250 rpm and 37 °C. Cry4AaCterGloverin IBs and Cry4AaCter-GFP IBs were also resolubilized in the same buffer at different pH values. For subsequent tag removal, the buffer was exchanged to thrombin buffer (20 mM Tris-HCl, 150 mM NaCl, 2.5 mM CaCl2, pH 8.4) using 5 ml PD MidiTrap G-25 gel-filtration columns (GE Healthcare, Little Chalfont, United Kingdom) in flowthrough mode. Samples collected in thrombin buffer were supplemented with 10 units of thrombin per milligram substrate protein and were incubated at room temperature for 24 h without shaking.

2.9.3. IMPI(I38V) activity assay IMPI(I38V) activity was detected with conducting a fluorescencebased activity assay [33] using the M4 metalloprotease thermolysin [34] and 1 μg IMPI(I38V) samples in TBS. We used 100 ng phosphoramidone as a positive control. The data are presented as means ± standard deviations of three experiments, with each result representing endpoints after 20 min substrate conversion. 2.9.4. Measurement of particle diameter The particle diameter distribution was measured by dynamic light scattering using a Mastersizer 3000 (Malvern Instruments, Malvern, United Kingdom). We added 2 ml of bacterial lysate to the measurement chamber filled with buffer A and took three measurements before calculating the results.

2.7. DSP of soluble GST-IMPI(I38V) The soluble fraction of the lysate was supplemented with 5 units of Benzonase (Merck) per gram wet weight of cell pellet used for lysis, and incubated in an ice bath on a magnetic stirrer at 150 rpm for 1 h. The soluble fraction was then passed through a 0.22-μm polyethersulfone (PES) bottle-top filter (Corning, New York, USA) and applied to a 5-ml glutathione affinity cartridge (Thermo Fisher Scientific, Waltham, USA) at a flow rate of 0.5 ml min−1 at room temperature using a NGC fast protein liquid chromatography (FPLC) system (Bio-Rad, Hercules, USA). The fusion protein was cleaved in situ by applying 40 units of thrombin per gram wet weight of the cell pellet used for lysis and incubating for 24 h at room temperature. IMPI(I38V) was washed from the column with buffer A, then diluted 1:25 with 10 mM sodium acetate (pH 4.0) and centrifuged (12000 x g, 10 min, 22 °C) to sediment flocculated particles. The supernatant was applied to a 4.7-ml HiScreen Capto SP ImpRes cation exchange column (GE Healthcare) at a flow rate of 1 ml min−1 using 10 mM sodium acetate (pH 4.0). Fractions were eluted in a 0–75% gradient of 1 M NaCl in 20 column volumes at room temperature. The fractions containing IMPI(I38V) were applied to a HiLoad 16/600 Superdex 75 pg size exclusion column (GE Healthcare) at a flow rate of 0.8 ml min−1 using Tris-buffered saline (TBS) (25 mM Tris-HCl, 150 mM NaCl, pH 7.4).

2.9.5. LPS assay The LPS content was determined using a fluorescence assay based on the LAL assay setup. Recombinant Factor C (Endozyme II, bioMérieux, Marcy-l`Etoile, France) was used according to the manufacturer's instructions. Glass vials were baked at 300 °C for 5 h and used for sample preparation. 2.9.6. Determination of thiol content To determine whether or not free thiol groups were present in the resolubilized IMPI(I38) samples, the Ellman assay was used [35]. A buffer exchange of protein samples was done with PD MidiTrap G-25 gel-filtration columns (GE Healthcare) to have all samples in Ellman reaction buffer (1 mM EDTA, 100 mM Na3PO4, pH 8.0). 50 μl of a 4 mg ml−1 5,5’-dithiobis-(2-nitrobenzoic acid) (Sigma-Aldrich) solution were mixed with 2.5 ml Ellman reaction buffer and 250 μl sample were added and incubated for 15 min at room temperature. Absorption of the samples at 412 nm was measured and the concentration of 2-nitro-5thiobenzoate was calculated with an extinction coefficient of 13 700 M−1 cm−1 and the sample dilution factor. Reduced glutathione (Merck, Darmstadt, Germany) was used as a standard in concentrations from 0.01 to 1.5 mM.

2.8. Precipitation of protein contaminants Contaminants in IMPI(I38V) samples after tag removal were precipitated with trichloroacetic acid (TCA) (Sigma-Aldrich) or heat [26]. For the TCA precipitation, samples were supplemented with 3% (w/v) ice-cold TCA and incubated on ice for 10 min before centrifugation (16000 x g, 10 min, 4 °C). The supernatant was transferred and neutralized with a volume of 2 M Tris equal to the volume of TCA added in the previous step. Heat precipitation was achieved by placing samples in a heat block at 100 °C for 10 min followed by centrifugation as above. The supernatant was transferred to a new reaction tube, and the buffer

2.9.7. GFP quantification The amount of fluorescent GFP after pH induced resolubilization of Cry4AaCter-GFP IBs was measured (GFP Quantitation Kit, Abnova, Taipei City, Taiwan) directly after resolubilization and buffer exchange of the samples in phosphate bufferd saline (PBS Dulbecco w/o Ca2+ w/ o Mg2+, Merck) using PD MidiTrap G-25 gel-filtration columns (GE 122

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Healthcare). An additional measurement of the same samples was done three days after the resolubilization. Samples had been stored in the dark at 8 °C. The measurement was done in white 96-well plates using an excitation wavelength of 485 nm and an emission wavelength of 528 nm.

resolubilization was only efficient with Cry4AaCter at the N-terminus, whereas IMPI(I38V)-Cry4AaCter was only sparingly soluble. This may reflect a general resolubilization effect on IBs in an alkaline buffer [37] but not the pH dependence of the Cry4AaCter tag. To exclude the possibility that freeze-thawing the IBs influenced the resolubilization process as previously described [38], a control sample of Cry4AaCterIMPI(I38V) IBs was treated with water instead of alkaline buffer after thawing and only marginal resolubilization was observed. We therefore concluded that for IMPI(I38V) pH dependent resolubilization was conferred by the N-terminal Cry4AaCter tag. As successful pH dependent resolubilization has been described for both N-terminal, and Cterminal orientation of the Cry4AaCter tag [20–23], a screening experiment for each new target protein is advisable.

2.9.8. Crossflow filtration Crossflow filtration was carried out at room temperature using a Sartoflow Slice 200 system and two Sartocon Slice 200 PESU membranes with pore sizes of 0.1 μm and 300 kDa, respectively (SartoriusStedim, Gottingen, Germany). The filtration area was 0.02 m2 in both cases. Filtration was performed with a constant transmembrane pressure (TMP) at 0.5 bar because good protein transmission at this TMP has already been reported with an IB-containing feed solution on a 0.1μm pore size membrane [36]. The system was equilibrated with 2 L buffer A with a TMP of 0.25 bar for the 0.1-μm membrane because without cake resistance the TMP of 0.5 bar could not be achieved. The feed solution was added to the feed tank in two consecutive 0.2-L steps with retentate and permeate valves completely open. Wash steps were similarly carried out using consecutive 0.2-L aliquots, requiring 0.4 L buffer A, 2 L 0.5% (v/v) Triton X-100, and 2 L Mili-Q water in the same process mode. Resolubilization with 0.4 L 50 mM NaHCO3 (pH 11.0) was carried out in recycle mode for 1 h. After the resolubilization step, the membrane was washed with 0.2 L resolubilization buffer in recycle mode for 30 min followed by a final wash with 0.2 L resolubilization buffer before all the valves were opened.

3.2. IMPI(I38V) activity assay To assess the activity of the recombinant IMPI(I38V) protein, the Cry4AaCter tag was proteolytically removed with thrombin after resolubilization and IMPI(I38V) was separated from the tag, the protease and residual impurities by precipitating the latter with 3% TCA or by heating to 100 °C. IMPI(I38V), with its stabilizing disulfide bonds, was expected to remain in the supernatant [26]. The recovered soluble IMPI (I38V) was then used in a fluorescence based activity assay (Fig. 2 A). The IMPI(I38V) sample produced as an insoluble Cry4AaCter fusion protein inhibited thermolysin. However, there was a difference in activity between samples treated with TCA and heat, with the former showing more activity even though the IBs in each case were derived from the same production batch. To determine whether this difference reflected a positive effect of TCA or a negative effect of heat, the experiment was repeated and TCA-treated samples were subsequently heated to 100 °C and the heat-treated samples were subsequently treated with TCA. We found that heating had no impact on the TCAtreated samples but that TCA increased the activity of the heat-treated samples (Fig. 2 B). It was also obvious that IMPI(I38V) treated with heat after tag removal displayed a variable quality, because the standard deviations in the inhibitory activity were high in the second experiment. The isolation of bioactive IMPI(I38V) therefore appeared to be partly dependent on the TCA precipitation step. A possible explanation is the effect of TCA on the stability of intermediate folding structures. For example, the 6.7 kDa peptide cardiotoxin III, which has disulfide bonds, was stabilized in the molten globule state by treatment with 3% TCA [39]. The molten globule state is a compact intermediate state with intact secondary structures that undergoes slow structural changes. The stable secondary structures may serve as starting point for the folding into a native tertiary structure [40]. A similar process could explain the more active structure of IMPI(I38V) molecules derived from TCA precipitation. If true, this would mean it is not necessary to use a mutant E. coli strain that forms disulfide bonds in the cytoplasm, because oxidation after TCA treatment would form the disulfide bonds in IMPI(I38V). Cry4AaCter-IMPI(I38V) was therefore produced in E. coli BL21(DE3) cells, and pH-sensitive IBs were formed as expected. After cleavage with thrombin and TCA precipitation, IMPI(I38V) was found in the soluble fraction and its bioactivity was confirmed (Fig. 3). In addition, an Ellman assay was used to screen for free thiol groups in the IMPI(I38V) samples and none were detected meaning that all cysteines formed disulfide bonds. We therefore proved that TCA treatment improves the structural stability of IMPI(I38V) produced as Cry4AaCter-induced IBs and allows the fusion protein to be produced in the standard BL21(DE3) strain. BL21(DE3) was chosen as the production strain for further experiments because this strain shows better growth characteristics than the mutant strain Rosetta-gami 2(DE3) pLysS. The cells were grown in a 5-L bioreactor in terrific broth at 37 °C in triplicate and we achieved a titer of 1665.7 ± 143.1 mg Cry4AaCter-IMPI(I38V) after high-pressure homogenization of the cells, corresponding to 512.5 ± 44 mg IMPI (I38V).

2.9.9. Microscopy Following high-pressure homogenization in buffer A, lysates were characterized by light microscopy using a DM750 microscope (Leica Microsystems, Wetzlar, Germany) at 1000-fold amplification. Images were captured using an integrated ICC50 camera (Leica Microsystems). Intact cells were analyzed by fluorescence microscopy using a DMI6000 microscope (Leica Microsystems). Cells were harvested after 4 h of production and the pelleted cells were stored at 8 °C overnight before resuspending them in PBS Instamed (Merck) for image acquisition the next morning. 3. Results and discussion 3.1. Tag orientation on IMPI(I38V) fusion proteins IMPI(I38V) fusion proteins with N-terminal or C-terminal Cry4AaCter tags were compared after producing both variants in E. coli Rosetta-gami™ 2(DE3) pLysS cells. Both variants, i.e. IMPI(I38V)Cry4AaCter and Cry4AaCter-IMPI(I38V), accumulated in the insoluble fraction after cell lysis showing that aggregation promoted by the tag was independent of its orientation (Fig. 1). However, alkaline

Fig. 1. SDS-PAGE analysis after the alkaline resolubilization of inclusion bodies (IBs). A, IMPI(I38V)-Cry4AaCter; B, Cry4AaCter-IMPI(I38V); control, IBs were thawed and treated with water instead of alkaline buffer; S, soluble fraction; I, insoluble fraction. Arrows mark the positions of the fusion proteins. 123

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Fig. 2. Fluorescence-based activity assay for IMPI(I38V) against thermolysin. Without inhibitor, buffer instead of inhibitor; phosphoramidone, positive control; rfu, relative fluorescence units. A IMPI(I38V) samples were treated with either heat or TCA for precipitating impurities. B IMPI(I38V) samples were treated first with either heat or TCA for precipitating impurities and second with the respective other method.

suitable membrane cut-off, we measured the particle size distribution of an IB sample by dynamic light scattering (Fig. 4). The range was 0.42–2.28 μm with the diameter of most particles between 0.78 and 1.24 μm (68.8% of all particles), which is consistent with IB size ranges in the literature [41]. Microscopy confirmed that the diameter of single particles was ∼1 μm, suggesting that the larger particles revealed by light scattering were probably aggregates. The nominal pore size of a membrane is an average of a pore size distribution, so it is advisable to use membranes with a cut-off that is 50% smaller than the smallest particle to be retained [42]. This indicated that our DSP strategy should include membranes with pores smaller than 0.45 μm. The size distribution of the particles after cell lysis (Fig. 4) showed only one peak, suggesting that the IBs and cell debris occupied a similar size range. We decided to use sodium tetraborate flocculation to separate the IBs from the debris because this compound interacts with carbohydrates and therefore could flocculate E. coli debris containing LPS. A similar method has been demonstrated for yeast [43]. Small aliquots of lysed E. coli cells with IBs were therefore mixed with sodium tetraborate and an endotoxin assay was used to detect LPS as a proxy for the success of cell debris removal. In 2-ml tubes, 94.4% of IBs could be recovered while the LPS content was reduced by 36.5%. Therefore, although flocculation reduced the LPS content (and presumable the cell debris), more efficient LPS reduction will be necessary to use IMPI(I38V) in a pharmaceutical context. We therefore compared several wash buffers for their ability to reduce the LPS content of the IB fraction compared to water as a control (Table 1). The best result was achieved by using 0.5% Triton X-100 as the wash buffer, and we therefore adopted this for our membrane-based DSP strategy for the recovery of IBs.

Fig. 3. Fluorescence-based activity assay for IMPI(I38V) against thermolysin. The Cry4AaCter-induced IBs were produced in E. coli BL21(DE3) and IMPI (I38V) was treated with 3% TCA after tag removal. Without inhibitor, buffer instead of inhibitor; phosphoramidone, positive control; rfu, relative fluorescence units.

3.3. Particle size distribution and cell debris removal We planned to use a crossflow membrane unit to develop a suitable DSP strategy for the Cry4AaCter-induced IBs because this allows the washing and resolubilization steps to be combined. To identify the most

Fig. 4. Distribution of the particle diameter of Cry4AaCter-IMPI(I38V) IBs after high pressure homogenization. The measurement was done by dynamic light scattering. The inset shows a microscopic control of the measurement. 124

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permeate after resolubilization relative to the content in the feed solution. LPS levels were reduced by 99.99% by both membranes indicating they were both suitable for efficient LPS removal. In conclusion, the membrane-based purification of Cry4AaCter-induced IBs was feasible with both membranes, but contaminant proteins were removed more efficiently by the 0.1 μm membrane. Furthermore, the flux at a constant TMP should remain higher with larger membrane pores, which would become relevant to avoid fouling when the process is scaled up.

Table 1 Evaluation of wash buffers for the reduction of LPS content in the IB fraction. EU, endotoxin units; EDTA, ethylenediaminetetraacetic acid. Sample

LPS content [EU ml−1]

IB fraction Wash buffer 1 mM EDTA 0.5% (v/v) Triton X-100 0.5% (v/v) TWEEN® 20 0.3 M arginine water

7.9 × 105 LPS content [EU ml−1] 3.7 × 104 1.1 × 103 1.3 × 105 9.4 × 104 7.4 × 104

3.5. Characterization of the membrane-based DSP strategy The new DSP strategy for the purification of Cry4AaCter-IMPI(I38V) IBs was carried out two further times using the 0.1 μm membrane in order to achieve a reliable characterization in terms of protein yield and LPS removal. Table 2 shows the means ± standard deviations of the three filtration experiments. The protein lost during the wash steps amounted to 17.8% of the starting amount, and an additional 14.9% of the target protein was lost during the membrane-based resolubilization step. The anticipated final yield in the permeate after resolubilization was ∼67.3% and the measured yield was very similar (69.2%) indicating that little or no protein adsorbed to the membrane material. The sodium borate flocculation step before membrane filtration was also examined because the earlier data were derived from small-scale experiments (2 ml) whereas flocculation prior to filtration was scaled up to 120 ml. The loss of target protein was 29.2% at the larger scale compared to 5.6% at the 2-ml scale. Moreover, the LPS reduction step was not effective: the LPS content relative to the lysate varied between −65.4% and +113.7%. According to the manufacturer of the LPS assay, a deviation of between 50% and 200% from control values in the assay is still regarded as a stable result because the assay system is prone to errors. However, in all the other experiments, despite high standard deviations, LPS reduction was confirmed. Thus, the sodium borate flocculation was not scalable from 2 ml to 120 ml and new empirical studies would be needed to find parameters suitable for larger feed volumes. Alternatively, the filtration step could be introduced directly after cell lysis to investigate whether an additional cell debris removal step prior to filtration is necessary. These modifications could increase the overall amount of target protein recovered after membrane-based purification because no protein would be lost during flocculation. The LPS content was successfully reduced during the course of filtration. Much of the LPS content was already removed by washing the IBs with buffer A. The Triton X-100 wash step did not result in the 99.99% LPS removal as in the membrane screening experiment, but it should be borne in mind that during the screening experiment the LPS content was also measured at the end, after the washing step with water. This indicates that Triton X-100 interferes with the hydrophobic interactions of LPS molecules and enables their removal in the subsequent washing step with water. Having confirmed that Cry4AaCter-IMPI(I38V) IBs can be washed and resolubilized by crossflow filtration combined with a simple pH shift, we tested the biological activity of the purified IMPI(I38V) target protein. We took 5 ml of resolubilized Cry4AaCter-IMPI(I38V) from each filtration and cleaved the fusion protein with thrombin to separate IMPI(I38V) from the tag and other impurities, which were removed by TCA precipitation as above. We then carried out the fluorescence-based activity assay using the soluble IMPI(I38V) fraction, and confirmed that the inhibitory activity of IMPI(I38V) was retained (Fig. 6).

3.4. Selection of a suitable membrane Three criteria were used to select a membrane for the purification of Cry4AaCter-induced IBs. First, the pore size must be less than 0.45 μm to ensure IBs remain in the retentate. Second, soluble protein contaminants and LPS should permeate the membrane so they can be removed by washing. Finally, because the purified IBs are resolubilized on the membrane, soluble Cry4AaCter-IMPI(I38V) should also permeate the membrane. Two Sartocon Slice 200 PESU membranes with a cut-off of 300 kDa or 0.1 μm were tested for this purpose. Fig. 5 shows SDS-PAGE analysis of samples from both filtration experiments. In both cases, IBs remained in the retentate during the washing steps with buffer A, 0.5% Triton X-100, and water, as indicated by the prominent ∼26 kDa band in the retentates and its much weaker counterpart in the permeates. Both membranes therefore satisfied the first selection criterion. The ∼35 kDa band in the permeate of the Triton X100 wash step was more prominent for the 0.1 μm membrane than the 300 kDa membrane, suggesting the former was advantageous in terms of contaminant protein permeability during the wash steps (criterion 2). Finally, the resolubilization of the IBs was possible with both membranes. The intensities of the bands in the retentate appear misleading, but the retentate volume was only 21 ml for the 300 kDa membrane and 25 ml for the 0.1 μm membrane, compared to a permeate volume of 752 ml for the 300 kDa membrane and 770 ml for the 0.1 μm membrane. The recovery of the target protein in the permeate was 70.3% with the 300 kDa membrane and 67.4% with the 0.1 μm membrane. Those values were comparable, and the purity of the target protein was therefore higher using the 0.1 μm membrane as seen in the samples of the permeate after resolubilization. To make a final decision on the most suitable membrane, we measured LPS levels in the

3.6. Comparison of soluble and insoluble production strategies

Fig. 5. SDS-PAGE analysis of samples taken from the retentates and permeates of consecutive process steps in two filtration experiments with different membrane cut-offs. M, Marker; feed, IB fraction in buffer A after sodium borate flocculation; R, retentate; P, permeate; buffer A, after washing with 0.4 L buffer A; Triton, after washing with 2 L 0.5% Triton X-100; water, after washing with 2 L Mili-Q H2O; resol, after resolubilization of IBs with 0.8 L 50 mM NaHCO3 buffer at pH 11.

GST-IMPI(I38V) was produced under the same conditions described above for Cry4AaCter-IMPI(I38V) but the E.coli strain Rosetta-gami 2(DE3) pLysS was used for production. IMPI is known to retain its biological activity when produced in this manner [34]. Although the recovery achieved in each DSP strategy was comparable, much more of 125

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Table 2 Purification yields of Cry4AaCter-IMPI(I38V) during the consecutive steps of the membrane-based DSP strategy. Protein yields were measured by SDS-PAGE and densitometry. LPS removal was measured in each fraction that contained the target protein. Values are means ± standard deviation of n = 3 experiments. % values were calculated with the mean + standard deviation of each step. n.a., not applicable; buffer A, after washing with 0.4 L buffer A; Triton, after washing with 2 L 0.5% Triton X-100; water, after washing with 2 L Mili-Q H2O; resolubilized, after resolubilization of the IBs with 0.8 L 50 mM NaHCO3 buffer at pH 11. Protein [mg] feed

Consecutive yields Stepwise loss

526 ± 84 100% / LPS reduction [EU/ml] feed

Consecutive reduction Stepwise reduction

1.19 × 107 ± 8.47 × 106 100% /

buffer A

Triton

water

resolubilized

permeate

permeate

permeate

retentate

permeate

8±2 −1.6% 1.6%

50 ± 3 −10.3% 8.8%

38 ± 8 −17.9% 8.4%

82 ± 9 −32.8% 18.2%

361 ± 61 69.2% /

buffer A retentate

Triton retentate

water retentate

resolubilized retentate

permeate

1.25 × 106 ± 6.75 × 105 90.55% 90.55%

3.1 × 105 ± 3.4 × 105 96.81% 66.23%

2.61 × 103 ± 1.41 × 103 99.98% 99.38%

n.a. n.a. /

46 ± 28.2 99.99% 99.98%

assay, but the LPS content of IMPI(I38V) samples after the membranebased DSP would also allow pharmaceutical experiments because a limit of 5 EU ml−1 kg−1 bodyweight is allowed for parenteral drugs [44] and the dose of IMPI(I38V) would probably be low because of its low IC50 [34]. These issues would not be relevant if IMPI(I38V) were developed for topical application. The DSP after soluble production took 7 days whereas the DSP after insoluble production was completed after 4.5 days. Overall, the IBbased approach is therefore more favorable for the production of IMPI (I38V). 3.7. Transferability of the IB based production To show that the Cry4AaCter-based pull-down and resolubilization approach can be used more generally for recombinant AMP production, the tag was also fused to the G. mellonella AMP gloverin for production using E. coli BL21(DE3) cells at the shaker-flask scale followed by alkaline resolubilization. Fig. 7 shows that the production as IBs was also successful with Cry4AaCter-Gloverin given that the ∼34 kDa target protein was exclusively present in the insoluble fraction of the lysate. The resolubilization step was most efficient at pH 11. In a further experiment, we investigated whether native structures in the protein fused to the Cry4AaCter tag remain intact in the IBs. This has not been investigated before because the proteins of interest fused to the tag displayed activity after DSP or after resolubilization in the guts of mosquito larvae [20,22,23]. The reporter protein GFP was

Fig. 6. Fluorescence-based activity assay for IMPI(I38V) against thermolysin after the DSP by crossflow filtration. IMPI(I38V) was treated with 3% TCA after tag removal. Without inhibitor, buffer instead of inhibitor; phosphoramidone, positive control; rfu, relative fluorescence units. Table 3 Purification yields of IMPI(I38V) after the soluble production of GST-IMPI (I38V) and the insoluble production of Cry4AaCter-IMPI(I38V) and the corresponding DSP strategies. The amount of fusion proteins was measured in the lysate by SDS-PAGE and densitometry. a The concentration of IMPI(I38V) after soluble production was only measured once after completion of three chromatography steps and tag removal. b The calculated value based on the results in Table 2, taking into account the sample dilution during tag removal, TCA precipitation and buffer exchange.

Fusion protein [μmol] IMPI(I38V) [μmol] Target protein yield [%] LPS content [EU ml−1]

Soluble production

Insoluble production

0.83 ± 0.27 0.12a 14.5 ± 3.6 0

61.7 ± 5.3 8.15 ± 1.37 13.2 ± 1 ∼ 16.7b

the fusion protein was produced when using the IB-based approach. Accordingly, the total yield of IMPI(I38V) after production as a soluble protein and purification by chromatography was 68-fold lower than with the IB-based approach followed by filtration-based DSP (Table 3). The LPS content of the IMPI(I38V) sample after DSP involving three chromatography steps was equal to the negative control in the LPS

Fig. 7. SDS-PAGE analysis of Cry4AaCter-gloverin production in BL21(DE3) cells, followed by resolubilization at different alkaline pH values. Lysate, after chemical cell lysis; resolubilization, after 1 h incubation in 50 mM NaHCO3 at various pH values and 37 °C. M, Marker; I, insoluble fraction; S, soluble fraction. 126

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Fig. 8. A Microscopic image of E. coli BL21(DE3) cells after the production of the Cry4AaCter-GFP fusion protein. Fluorescent inclusion bodies are shown with arrows. B SDS-PAGE analysis of Cry4AaCter-GFP production in BL21(DE3) cells, followed by resolubilization at different alkaline pH values. Lysate, after chemical cell lysis; resolubilization, after 1 h incubation in 50 mM NaHCO3 at various pH values and 37 °C. M, Marker; I, insoluble fraction; S, soluble fraction.

therefore fused to the Cry4AaCter tag and the fusion protein was expressed in E. coli BL21(DE3). Fig. 8 A shows a merged fluorescence microscopy image showing GFP fluorescence in the IBs, thus confirming that the protein of interest can achieve its native conformation in the context of Cry4AaCter-induced IBs. The fluorescence of GFP fused to pull-down tags has previously been used to determine whether native protein structures persist in IBs [45–51]. Fig. 8 B prooves that the production of Cry4AaCter-GFP with a molecular weight of approximately 43 kDa resulted exclusively in insoluble protein. The resolubilization at alkaline pH was successful and most efficient at pH 11 (Fig. 8 B). From this and the resolubilization results with Cry4AaCtergloverin (Fig. 7) we conclude that the resolubilization pH for the best recovery of the produced fusion protein is independent of the target protein fused to the Cry4AaCter tag. The results with Cry4AaCter as a pull-down tag indicate that protein structure within the IBs is variable in quality, given that not all IBs showed fluorescence and the fluorescence intensity was variable among different IBs (Fig. 8A). After resolubilization of Cry4AaCter-GFP at different pH values the amount of fluorescent GFP was quantified in the resolubilized samples (Fig. 9) to determine whether or not native structures of the protein of interest endure the alkaline resolubilization. GFP fluorescence was detected in all samples and the highest total amount of native GFP was detected in the sample resolubilized at pH 11. Moreover, it was shown,

that after storage of Cry4AaCter-GFP in PBS buffer for three days at 8 °C, refolding of GFP took place because the GFP fluorescence increased. Relating the content of fluorescent GFP to the content of GFP in the respective Cry4AaCter-GFP fusion protein samples (Table 4) showed that only very few GFP molecules had a native structure after resolubilization, but the highest content of native GFP was found after resolubilization at pH 10. These findings show that native structures of target proteins can endure the alkaline resolubilization. However, optimization of the resolubilization process is necessary. For example, resolubilization at less alkaline conditions for longer periods of time could help recovering more native structures. The production conditions could also be refined so that the content of native structures in Cry4AaCter-induced IBs would increase. Producing the IBs at 30 °C instead of 37 °C resulted in higher activity of the fusion protein in another study [23]. A more sophisticated approach would be the creation of a synthetic pull-down tag based on the conserved block 7 sequences of the Cry4Aa toxin which is most efficient for the pull-down and Cry1Aa toxin which is most efficient for preserving bioactivity of the fusion protein [52]. This protein design approach together with finding and controlling the parameters that determine the protein quality in Cry4AaCter-induced IBs would offer the prospect of producing diverse recombinant proteins using this straightforward IB-based strategy. Replacing the proteolytical tag removal with chemical cleavage, which is suitable for many AMPs [53], could reduce the DSP cost and make this process even more attractive. The membrane based DSP could then also be established for the separation of the tag and the target protein to accomplish an additional process step in a single membrane unit. 4. Conclusion We have shown that the proteins of interest fused to the Cry4AaCter tag can retain their native characteristics in Cry4AaCter-induced IBs and the simple pH-dependent resolubilization step was independent of Table 4 Relative content of native GFP in 100 μl Cry4AaCter-GFP samples resolubilized at different pH values. The total amount of the fusion protein was measured using densitometric band analysis in a SDS-PAGE with bovine serum albumin as standard. The amount of fluorescent GFP measured in each sample (Fig. 9) relative to the total amount of GFP is shown.

pH pH pH pH

Fig. 9. Quantification of fluorescent GFP directly after resolubilization of Cry4AaCter-GFP IBs at different alkaline pH values and after storing the resolubilized Cry4AaCter-GFP samples for three days (3 d) in PBS at 8 °C in the dark. 127

8 9 10 11

Fusion protein [μg]

native GFP after resolubilization [%]

native GFP 3 d after resolubilization [%]

20.35 20.93 43.68 261.63

0.29 0.49 0.76 0.43

0.77 1.43 4.82 2.78

± ± ± ±

0.05 0.05 0.03 0.04

± ± ± ±

0.06 0.05 0.33 0.42

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the protein of interest fused to the tag in the three cases we investigated. A crossflow filtration DSP strategy for Cry4AaCter-induced IBs was established and both washing and resolubilization could be performed in a single process unit. IMPI(I38V), a protease inhibitor of pharmaceutical interest, was isolated in a biologically active form after the membrane-based purification of Cry4AaCter-induced IBs and the LPS content of the target protein was reduced to a level that enables pharmaceutical testing. The native conformation of GFP inside Cry4AaCter-induced IBs shows that the pull-down tag could be generally useful for the production of recombinant proteins and not only for the production of AMPs. The suitability of this approach for AMP production was confirmed by the successful production and resolubilization of gloverin fused to the Cry4AaCter tag. The Cry4AaCter tag and membrane-based DSP therefore offer a versatile platform technology for the production of recombinant AMPs and other proteins in E. coli.

1568026616666160713123654. [13] M. Tonk, A. Vilcinskas, M. Rahnamaeian, Insect antimicrobial peptides. Potential tools for the prevention of skin cancer, Appl. Microbiol. Biotechnol. 100 (17) (2016) 7397–7405, https://doi.org/10.1007/s00253-016-7718-y. [14] J. Zitzmann, T. Weidner, G. Eichner, et al., Dielectric spectroscopy and optical density measurement for the online monitoring and control of recombinant protein production in stably transformed Drosophila melanogaster S2 cells, Sensors 18 (3) (2018), https://doi.org/10.3390/s18030900. [15] M. Berkmen, Production of disulfide-bonded proteins in Escherichia coli, Protein Expr. Purif. 82 (1) (2012) 240–251, https://doi.org/10.1016/j.pep.2011.10.009. [16] C. Walther, M. Kellner, M. Berkemeyer, et al., Integrated process development-a robust, rapid method for inclusion body harvesting and processing at the microscale level, Prep. Biochem. Biotechnol. 47 (9) (2017) 874–880, https://doi.org/10.1080/ 10826068.2017.1350978. [17] R.V. Datar, T. Cartwright, C.G. Rosen, Process economics of animal cell and bacterial fermentations: a case study analysis of tissue plasminogen activator, Biotechnology (N Y) 11 (3) (1993) 349–357. [18] Z. Lin, Q. Zhao, L. Xing, et al., Aggregating tags for column-free protein purification, Biotechnol. J. 10 (12) (2015) 1877–1886, https://doi.org/10.1002/biot. 201500299. [19] D. Hoffmann, M. Ebrahimi, D. Gerlach, et al., Reassessment of inclusion body-based production as a versatile opportunity for difficult-to-express recombinant proteins, Crit. Rev. Biotechnol. 38 (5) (2018) 729–744, https://doi.org/10.1080/07388551. 2017.1398134. [20] T. Hayakawa, S. Sato, S. Iwamoto, et al., Novel strategy for protein production using a peptide tag derived from Bacillus thuringiensis Cry4Aa, FEBS J. 277 (13) (2010) 2883–2891, https://doi.org/10.1111/j.1742-4658.2010.07704.x. [21] M. Hayashi, S. Iwamoto, S. Sato, et al., Efficient production of recombinant cystatin C using a peptide-tag, 4AaCter, that facilitates formation of insoluble protein inclusion bodies in Escherichia coli, Protein Expr. Purif. 88 (2) (2013) 230–234, https://doi.org/10.1016/j.pep.2013.01.011. [22] R. Matsumoto, Y. Shimizu, M.T.H. Howlader, et al., Potency of insect-specific scorpion toxins on mosquito control using Bacillus thuringiensis Cry4Aa, J. Biosci. Bioeng. 117 (6) (2014) 680–683, https://doi.org/10.1016/j.jbiosc.2013.12.004. [23] T. Okazaki, J. Ichinose, S. Takebe, et al., Potency of the mosquitocidal Cry46Ab toxin produced using a 4AaCter-tag, which facilitates formation of protein inclusion bodies in Escherichia coli, Appl. Entomol. Zool. 177 (2017) 6027, https://doi.org/ 10.1007/s13355-017-0529-5. [24] K.F. Kane, D.L. Hartley, Formation of recombinant protein inclusion bodies in Escherichia coli, TIBTECH 6 (1988) 95–101. [25] E. Ledung, P.-O. Eriksson, S. Oscarsson, A strategic crossflow filtration methodology for the initial purification of promegapoietin from inclusion bodies, J. Biotechnol. 141 (1–2) (2009) 64–72, https://doi.org/10.1016/j.jbiotec.2009.02.016. [26] M. Wedde, C. Weise, P. Kopacek, et al., Purification and characterization of an inducible metalloprotease inhibitor from the hemolymph of greater wax moth larvae, Galleria mellonella, Eur. J. Biochem. 255 (3) (1998) 535–543, https://doi. org/10.1046/j.1432-1327.1998.2550535.x. [27] J.L. Arolas, T.O. Botelho, A. Vilcinskas, et al., Structural evidence for standardmechanism inhibition in metallopeptidases from a complex poised to resynthesize a peptide bond, Angew. Chem. 50 (44) (2011) 10357–10360, https://doi.org/10. 1002/anie.201103262. [28] O.A. Adekoya, I. Sylte, The thermolysin family (M4) of enzymes. Therapeutic and biotechnological potential, Chem. Biol. Drug Des. 73 (1) (2009) 7–16, https://doi. org/10.1111/j.1747-0285.2008.00757.x. [29] C. Schreiber, H. Müller, O. Birrenbach, et al., A high-throughput expression screening platform to optimize the production of antimicrobial peptides, Microb. Cell Factories 16 (1) (2017) 29, https://doi.org/10.1186/s12934-017-0637-5. [30] U. Ritzén, J. Rotticci-Mulder, P. Strömberg, et al., Endotoxin reduction in monoclonal antibody preparations using arginine, J. Chromatogr. B. Anal. Technol. Biomed. Life Sci. 856 (1–2) (2007) 343–347, https://doi.org/10.1016/j.jchromb. 2007.06.020. [31] M.M. Weiser, L. Rothfield, The reassociation of lipopolysaccharide, phospholipid, and transferase enzymes of the bacterial cell envelope. Isolation of binary and ternary complexes, J. Biol. Chem. 243 (6) (1968) 1320–1328. [32] H. Jang, H.-S. Kim, S.-C. Moon, et al., Effects of protein concentration and detergent on endotoxin reduction by ultrafiltration, BMB Rep. 42 (7) (2009) 462–466. [33] S.S. Twining, Fluorescein isothiocyanate-labeled casein assay for proteolytic enzymes, Anal. Biochem. 143 (1) (1984) 30–34, https://doi.org/10.1016/00032697(84)90553-0. [34] M. Eisenhardt, D. Dobler, P. Schlupp, et al., Development of an insect metalloproteinase inhibitor drug Carrier system for application in chronic wound infections, J. Pharm. Pharmacol. (2015), https://doi.org/10.1111/jphp.12452. [35] G.L. Ellman, Tissue sulfhydryl groups, Arch. Biochem. Biophys. 82 (1) (1959) 70–77, https://doi.org/10.1016/0003-9861(59)90090-6. [36] S.M. Bailey, M.M. Meagher, Crossflow microfiltration of recombinant Escherichia coli lysates after high pressure homogenization, Biotechnol. Bioeng. 56 (3) (1997) 304–310, https://doi.org/10.1002/(SICI)1097-0290(19971105)56 3 < 304:AIDBIT8 > 3.0.CO;2-N. [37] J.T. Heiker, N. Kloting, M. Bluher, et al., Access to gram scale amounts of functional globular adiponectin from E. coli inclusion bodies by alkaline-shock solubilization, Biochem. Biophys. Res. Commun. 398 (1) (2010) 32–37, https://doi.org/10.1016/j. bbrc.2010.06.020. [38] X. Qi, Y. Sun, S. Xiong, A single freeze-thawing cycle for highly efficient solubilization of inclusion body proteins and its refolding into bioactive form, Microb. Cell Factories 14 (2015) 24, https://doi.org/10.1186/s12934-015-0208-6. [39] T. Sivaraman, T.K. Kumar, G. Jayaraman, et al., The mechanism of 2,2,2-

Declarations of interest None. Funding This work was financially supported by the Hessian Ministry of Education and Art within the Hessian initiative for supporting economic and scientific excellence (LOEWE). Part of the work was funded by the German Ministery of Education and Research (BMBF) via the project 4-IN. Acknowledgements We acknowledge Dr. Richard M. Twyman for editorial assistance. References [1] E.Y. Klein, T.P. van Boeckel, E.M. Martinez, et al., Global increase and geographic convergence in antibiotic consumption between 2000 and 2015, Proc. Natl. Acad. Sci. U. S. A. 5 (2018), https://doi.org/10.1073/pnas.1717295115. [2] H. Goossens, M. Ferech, R. Vander Stichele, et al., Outpatient antibiotic use in Europe and association with resistance. A cross-national database study, Lancet 365 (9459) (2005) 579–587, https://doi.org/10.1016/S0140-6736(05)17907-0. [3] D.I. Andersson, D. Hughes, J.Z. Kubicek-Sutherland, Mechanisms and consequences of bacterial resistance to antimicrobial peptides, Drug Resist. Updates : Rev. Comment. Antimicrob. Anticancer Chemother. 26 (2016) 43–57, https://doi.org/ 10.1016/j.drup.2016.04.002. [4] M.R. Bolouri Moghaddam, M. Tonk, C. Schreiber, et al., The potential of the Galleria mellonella innate immune system is maximized by the co-presentation of diverse antimicrobial peptides, Biol. Chem. 397 (9) (2016) 939–945, https://doi. org/10.1515/hsz-2016-0157. [5] A.-K. Pöppel, H. Vogel, J. Wiesner, et al., Antimicrobial peptides expressed in medicinal maggots of the blow fly Lucilia sericata show combinatorial activity against bacteria, Antimicrob. Agents Chemother. 59 (5) (2015) 2508–2514, https:// doi.org/10.1128/AAC.05180-14. [6] H.-K. Kang, C. Kim, C.H. Seo, et al., The therapeutic applications of antimicrobial peptides (AMPs). A patent review, J. Microbiol. (Seoul, Korea) 55 (1) (2017) 1–12, https://doi.org/10.1007/s12275-017-6452-1. [7] Z. Zheng, N. Tharmalingam, Q. Liu, et al., Synergistic efficacy of Aedes aegypti antimicrobial peptide cecropin A2 and tetracycline against Pseudomonas aeruginosa, Antimicrob. Agents Chemother. 61 (7) (2017), https://doi.org/10.1128/AAC. 00686-17. [8] Y. Briers, M. Walmagh, B. Grymonprez, et al., Art-175 is a highly efficient antibacterial against multidrug-resistant strains and persisters of Pseudomonas aeruginosa, Antimicrob. Agents Chemother. 58 (7) (2014) 3774–3784, https://doi.org/ 10.1128/AAC.02668-14. [9] Y. Briers, M. Walmagh, V. van Puyenbroeck, et al., Engineered endolysin-based "Artilysins" to combat multidrug-resistant gram-negative pathogens, mBio 5 (4) (2014) e01379-14, , https://doi.org/10.1128/mBio.01379-14. [10] V. Defraine, J. Schuermans, B. Grymonprez, et al., Efficacy of artilysin art-175 against resistant and persistent acinetobacter baumannii, Antimicrob. Agents Chemother. 60 (6) (2016) 3480–3488, https://doi.org/10.1128/AAC.00285-16. [11] E. Mylonakis, L. Podsiadlowski, M. Muhammed, et al., Diversity, evolution and medical applications of insect antimicrobial peptides, Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 371 (1695) (2016), https://doi.org/10.1098/rstb.2015.0290. [12] M. Tonk, A. Vilcinskas, The medical potential of antimicrobial peptides from insects, CTMC 17 (5) (2016) 554–575, https://doi.org/10.2174/

128

Protein Expression and Purification 155 (2019) 120–129

D. Hoffmann et al.

[40] [41] [42] [43] [44] [45]

[46] [47]

trichloroacetic acid-induced protein precipitation, J. Protein Chem. 16 (4) (1997) 291–297. K. Kuwajima, The molten globule state as a clue for understanding the folding and cooperativity of globular-protein structure, Proteins 6 (2) (1989) 87–103, https:// doi.org/10.1002/prot.340060202. G. Taylor, M. Hoare, Gray, et al., Size and density of protein inclusion bodies, Nat. Biotechnol. 4 (6) (1986) 553–557. T. Melin, R. Rautenbach, Membranverfahren. Grundlagen der Modul- und Anlagenauslegung, 3., aktualisierte und erweiterte Auflage. VDI-Buch, SpringerVerlag, Berlin und Heidelberg, 2007. J. Bonnerjea, J. Jackson, M. Hoare, et al., Affinity flocculation of yeast cell debris by carbohydrate-specific compounds, Enzym. Microb. Technol. 10 (6) (1988) 357–360, https://doi.org/10.1016/0141-0229(88)90015-4. P. Malyala, M. Singh, Endotoxin limits in formulations for preclinical research, J. Pharmaceut. Sci. 97 (6) (2008) 2041–2044, https://doi.org/10.1002/jps.21152. E. García-Fruitós, N. González-Montalbán, M. Morell, et al., Aggregation as bacterial inclusion bodies does not imply inactivation of enzymes and fluorescent proteins, Microb. Cell Factories 4 (2005) 27, https://doi.org/10.1186/1475-28594-27. M. Morell, R. Bravo, A. Espargaró, et al., Inclusion bodies: specificity in their aggregation process and amyloid-like structure, Biochim. Biophys. Acta 1783 (10) (2008) 1815–1825, https://doi.org/10.1016/j.bbamcr.2008.06.007. N.S. de Groot, F.X. Aviles, J. Vendrell, et al., Mutagenesis of the central

[48] [49] [50] [51] [52]

[53]

129

hydrophobic cluster in Abeta42 Alzheimer's peptide. Side-chain properties correlate with aggregation propensities, FEBS J. 273 (3) (2006) 658–668, https://doi.org/10. 1111/j.1742-4658.2005.05102.x. W. Kim, M.H. Hecht, Sequence determinants of enhanced amyloidogenicity of Alzheimer A{beta}42 peptide relative to A{beta}40, J. Biol. Chem. 280 (41) (2005) 35069–35076, https://doi.org/10.1074/jbc.M505763200. B. Zhou, L. Xing, W. Wu, et al., Small surfactant-like peptides can drive soluble proteins into active aggregates, Microb. Cell Factories 11 (2012) 10, https://doi. org/10.1186/1475-2859-11-10. W. Wu, L. Xing, B. Zhou, et al., Active protein aggregates induced by terminally attached self-assembling peptide ELK16 in Escherichia coli, Microb. Cell Factories 10 (2011) 9, https://doi.org/10.1186/1475-2859-10-9. Z. Lin, B. Zhou, W. Wu, et al., Self-assembling amphipathic alpha-helical peptides induce the formation of active protein aggregates in vivo, Faraday Discuss 166 (2013) 243, https://doi.org/10.1039/c3fd00068k. T. Hayakawa, Y. Shimizu, T. Ishida, et al., Mutational analyses of Cry protein block7 polypeptides that facilitate the formation of protein inclusion in Escherichia coli, Appl. Microbiol. Biotechnol. 90 (6) (2011) 1943–1951, https://doi.org/10.1007/ s00253-011-3216-4. Y. Li, Z. Chen, RAPD. A database of recombinantly-produced antimicrobial peptides, FEMS Microbiol. Lett. 289 (2) (2008) 126–129, https://doi.org/10.1111/j. 1574-6968.2008.01357.x.