BRIEF RESEARCH REPORT published: 29 April 2022 doi: 10.3389/fbioe.2022.842256 Toxicity Profiling of Bacterial Inclusion Bodies in Human Caco-2 Cells Irene Barguilla 1, Ugutz Unzueta 1,2,3,4, Jose Vicente Carratalá 4,5, Olivia Cano-Garrido 4,5, Antonio Villaverde 1,4,5, Alba Hernández 1,6* and Neus Ferrer-Miralles 1,4,5* 1 Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, Spain, 2Biomedical Research Institute Sant Pau (IIB Sant Pau), Barcelona, Spain, 3Josep Carreras Leukaemia Research Institute, Barcelona, Spain, 4 Networking Center for Biomedical Research in Bioengineering, Biomaterials, and Nanomedicine (CIBER-BBN), Carlos III Institute of Health, Madrid, Spain, 5Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, Spain, 6 Consortium for Biomedical Research in Epidemiology and Public Health (CIBERESP), Carlos III Institute of Health, Madrid, Spain Bacterial inclusion bodies (IBs) are discrete macromolecular complexes that appear in recombinant prokaryotic cells under stress conditions. These structures are often discarded for biotechnological uses given the difficulty in recovering proteins of interest from them in a soluble form. However, recent approaches have revealed the potential of these protein clusters as biomaterials to promote cell growth and as protein depots for the Edited by: release of recombinant proteins for biotechnological and biomedical applications. Sílvia Pujals, Although these kinds of natural supramolecular complexes have attracted great Institute for Bioengineering of Catalonia (IBEC), Spain interest, no comprehensive study of their toxicity in cell cultures has been carried out. Reviewed by: In this study, caco-2 cells were exposed to natural IBs, soluble protein-only nanoparticles Georgios Skretas, (NPs), and non-assembled versions of the same protein for comparative purposes. National Hellenic Research Foundation, Greece Cytotoxicity, oxidative stress, and genotoxicity were analyzed for all these protein Artur Ribeiro, formats. Natural IBs and soluble protein formats demonstrated their safety in University of Minho, Portugal eukaryotic cells. No cytotoxicity, genotoxicity, or oxidative stress was detected in *Correspondence: caco-2 cells exposed to the protein samples in any of the experimental conditions Neus Ferrer-Miralles neus.ferrer@uab.cat evaluated, which covered protein concentrations used in previous biological activity Alba Hernández assays. These conditions evaluated the activity of protein samples obtained from three alba.hernandez@uab.cat prokaryotic hosts [Escherichia coli and the endotoxin-free expression systems Specialty section: Lactococcus lactis and ClearColi® BL21 (DE3)]. Our results demonstrate that natural This article was submitted to IBs and soluble protein nanoparticles are non-toxic materials for eukaryotic cells and that Nanobiotechnology, this may represent an interesting alternative to the classical unassembled format of a section of the journal Frontiers in Bioengineering and recombinant proteins for certain applications in biotechnology and biomedicine. Biotechnology Keywords: inclusion bodies, cytotoxicity, genotoxicity, recombinant protein, caco-2 cells Received: 23 December 2021 Accepted: 21 March 2022 Published: 29 April 2022 INTRODUCTION Citation: Barguilla I, Unzueta U, Carratalá JV, The global biopharmaceutical market is in expansion and actively seeking novel biomaterials to cover Cano-Garrido O, Villaverde A, the unmet needs of biomedical and biotechnological applications (Fenton et al., 2018; Puertas- Hernández A and Ferrer-Miralles N Bartolomé et al., 2021). One type of complex biological product that has invaded the biopharmaceutical (2022) Toxicity Profiling of Bacterial Inclusion Bodies in Human Caco- market in the recent decades is recombinant proteins, and among them, monoclonal antibodies 2 Cells. (mAbs) are blockbuster drugs (Ecker et al., 2015; Sanchez-Garcia et al., 2016). In most of the products Front. Bioeng. Biotechnol. 10:842256. approved for clinical uses, the protein of interest is administered by the parenteral route in a soluble doi: 10.3389/fbioe.2022.842256 form (Ibraheem et al., 2014). Unfortunately, owing to the pharmacokinetic characteristics of antibodies Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 1 April 2022 | Volume 10 | Article 842256 Barguilla et al. Profiling Cytotoxicity of Inclusion Bodies and recombinant proteins in general, a significant portion of the the European Food Safety Authority (EFSA) and considered protein does not reach the biological target (Petitcollin et al., 2020). Generally Recognized as Safe (GRAS) by the U.S. Food and Strategies to cope with this reduced effective dose are being Drug Administration (FDA) (Morello et al., 2008; Gifre-Renom developed, such as the so-called virus-like particles (VLPs) or et al., 2018). Therefore, the effect of the presence of endotoxins in soluble protein nanoparticles (NPs). These particles are formed prokaryotic systems needs to be addressed and these contaminants by higher-order complexes of several copies of the same need to be eliminated from the final product. In this work, the recombinant protein, which can mitigate the renal clearance toxicity of the protein aggregates, known in prokaryotic hosts as effect but can be concomitantly associated with the formation inclusion bodies (IBs), was analyzed at the cellular level, taking into of protein corona and stimulation of cellular uptake by phagocytic consideration the cytotoxicity, genotoxicity, and oxidative stress of and non-phagocytic cells (Cheng et al., 2015). the interaction of such protein formats with cultured cells. Two In this regard, the formation of protein supramolecular unrelated His-tagged recombinant proteins which can be produced complexes in nature is not an isolated event. In fact, in the and purified from the soluble and insoluble cell fractions were recombinant protein area, the presence of protein aggregates has selected: T22-GFP-H6, a GFP-containing protein that carries an been widely documented in all expression systems tested so far N-terminal domain with the ability to bind to CXCR4 receptor (Kopito, 2000; Rodríguez-Carmona et al., 2015; Rueda et al., 2016; (Unzueta et al., 2017), and IFN-γ-H6, a pleiotropic Gifre-Renom et al., 2018; De Marco et al., 2019). At the immunomodulator (Schroder et al., 2004). These analysis were ultrastructural level, these protein aggregates are stable structures compared with two soluble recombinant proteins formats including with a basic scaffold formed by intramolecular and intermolecular NPs (Unzueta et al., 2012; López-Laguna et al., 2019) as well as non- interactions between beta sheets of the same recombinant protein assembled protein counterparts from protein samples obtained (De Marco et al., 2019). Embedded in this cage, recombinant from endotoxin-producing cells as well as endotoxin-free protein conformers, corresponding to a mixture of native and prokaryotic expression systems (Supplementary Figure S1). non-native protein species, can be released by physical means including complete denaturing processes up to solubilization with mild detergents under non-denaturing conditions (Carratalá METHODS et al., 2021). In any case, the evidence supporting the presence of biological activity among the protein subpopulations forming part Bacterial Strains and Plasmids of these aggregates has completely changed the view of this type of A synthetic gene encoding the modular protein T22-GFP-H6 was biological material from waste to reusable biocompatible material. designed in house, produced by Geneart (Thermo Fisher Scientific, Initial studies were directed toward the analysis of enzyme Waltham, MA, United States) and subcloned into the expression immobilization in repeated cycles for industrial applications vector pET22b (Novagen, Merck KGaA, Darmstadt, Germany). (Jäger et al., 2020). Other types of approaches have explored the T22 is a chemokine receptor (CXCR4) antagonist derived from the biocompatible nature of these protein clusters in tissue regeneration polyphemousin II that has an antiparallel β sheet structure approaches (Díez-Gil et al., 2010; Seras-Franzoso et al., 2012; stabilized by two disulfide bonds (Murakami et al., 1997). E. coli Tatkiewicz et al., 2019) or even as protein depots of releasable Origami B (Novagen) was used as expression system for the T22- active protein with tumor-targeting potential in animal models GFP-H6 formats as shown in Table 1 (Protein NPs and IBs). In (Pesarrodona et al., 2019; Céspedes et al., 2020). With all these data this E. coli strain, the thioredoxin reductase (trxB) and glutathione taken together, IBs are envisioned as a novel biotechnological reductase (gor) genes are deleted, facilitating disulfide bond platform of nanomaterial based on recombinant proteins. formation (Xu et al., 2008). A synthetic gene encoding IFN-γ- However, despite being a source of biological active protein as H6 was designed in house and produced by Geneart (Thermo well as a nanomaterial with intrinsic biomedical potential, detailed Fisher Scientific) as a codon optimized version for Lactococcus analysis of the toxicity of this type of protein clusters has not been lactis host and subcloned into pETDuet (Novagen) for E. coli fully explored. The presence of endotoxins in recombinant proteins ® expression system and pNZ8148 plasmid (Cm , MoBiTec GmbH, produced in E. coli has been widely described (Magalhães et al., 2007; Mamat et al., 2015). However, this type of compounds can be Göttingen, Germany) for L. lactis expression system. ClearColi BL21 (DE3) strain (Lucigen, Middleton, WI, United States) was ® removed from the purified protein by established methods, which used as expression system for IFN-γ-H6 protein formats have allowed biopharmaceutical products on the market to be (unassembled protein format and IBs) in an E. coli endotoxin- approved, insulin being the paradigmatic example of this free strain (Table 1). IFN-γ-H6 was also produced in Lactococcus expression system (Ferrer-Miralles et al., 2009; Baeshen et al., 2015). In parallel, other prokaryotic expression systems are being ® lactis expression strain NZ9000 (NICE , Boca Scientific, Inc., Dedham, MA, United States) (Mireau 2005 AMB) in the same developed to produce recombinant proteins lacking these specific toxins. An E. coli-derived mutant strain has been modified to inhibit ® protein formats as in ClearColi (Table 1). the synthesis route of the lipopolysaccharide of E. coli and is now Soluble Protein Production and Purification ® commercially available (ClearColi BL21 (DE3)) (Planesse et al., 2015; Viranaicken et al., 2017). Another strategy is to use Gram- of Soluble Protein Formats positive microorganisms that do not synthesize endotoxins as pET22b-T22-GFP-H6 plasmid was transformed into E. coli Gram-negatives do. An interesting alternative is the Gram- Origami B (Novagen) by heat shock at 42°C and maintained positive Lactococcus lactis, which is an accepted food additive by in lysogenic broth (LB) supplemented with 100 μg/ml ampicillin. Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 2 April 2022 | Volume 10 | Article 842256 Barguilla et al. Profiling Cytotoxicity of Inclusion Bodies TABLE 1 | Detailed information of the protein name, cell fraction origin, protein format, and expression system of the recombinant proteins used in the study. Sample # Protein name/MW Cell fraction Protein format Expression vector Expression system 1 IFN-γ-H6 / 18.02 ICF IBs pETDuet Clearcoli® 2 IFN-γ-H6 / 18.02 SCF unassembled pETDuet Clearcoli® 3 IFN-γ-H6 / 18.02 ICF IBs pNZ8148 L. lactis NZ9000 4 IFN-γ-H6 / 18.02 SCF unassembled pNZ8148 L. lactis NZ9000 5 T22-GFP-H6 / 30.7 ICF IBs pET22b E. coli Origami B 6 T22-GFP-H6 / 30.7 SCF Protein NPs pET22b E. coli Origami B Each protein was analyzed in two different formats as indicated. ICF, insoluble cell fraction; SCF, soluble cell fraction. Three protein formats were obtained: IBs, inclusion bodies; protein NPs, protein nanoparticles and unassembled soluble protein. MW, molecular weight in kDa. Recombinant protein was produced in the same medium O/N at adding 500 ng/ml nisin (Sigma–Aldrich). After induction, 20°C upon induction with 0.1 mmol/L isopropyl- β-D-1- cultures were grown for 3 h. Bacteria were harvested at 5,000 g thiogalactopyranoside (IPTG; Sigma–Aldrich, Sant Louis, MO, for 5 min at 4°C and washed twice with PBS. IFN-γ-H6 was United States). Cells were then harvested by centrifugation purified from the soluble cell fraction by affinity chromatography (10 min at 5,000 g). T22-GFP-H6 was purified from the (Supplementary Method). Protein purity was determined by soluble cell fraction by affinity chromatography SDS-PAGE gel electrophoresis and final protein concentration (Supplementary Method). Protein purity was determined by was determined by Bradford assay. SDS-PAGE gel electrophoresis and subsequent protein Different protein formats of T22-GFP-H6 and IFN-γ-H6 immunodetection by Western blot using an anti-His obtained from the soluble cells fractions of the expression monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, hosts are represented in Supplementary Figure S1. United States; ref: Sc-57598). Protein integrity was then verified by MALDI-TOF mass spectrophotometry in a linear mode (20–80 kDa). For that, 5 µl of protein sample was first Insoluble Protein Production and dialyzed against milli-Q H2O in a Millipore membrane for 20min Purification of IBs and loaded then onto a MALDI plate with Siapinic Acid in pET22b-T22-GFP-H6 plasmid was transformed into E. coli sandwich mode. Final protein concentration was determined by Origami B (Novagen) by heat shock at 42°C and maintained Bradford assay. Formation of protein NPs (generated by the self- in LB supplememted with 100 μg/ml ampicillin. Recombinant assembling of monomeric building blocks of recombinant protein was produced in the same medium for 3 h at 37°C upon protein) was determined by dynamic light scanning (DLS) induction with 1 mmol/L isopropyl- β-D-1-thiogalactopyranoside analysis in a Zetasizer Nano ZS (Malvern panalytical, Malvern, (IPTG). Cells from 20 ml of culture were then harvested by United Kingdom). The factors involved in the formation of centrifugation (5 min at 5,000 g). Purification of IBs of T22- protein NPs from soluble recombinant proteins have been GFP-H6 was performed following protocol described in previously described and seem to be related to the cationic Supplementary Method. Protein purity was determined by nature of the peptide located at the N-terminal end of the SDS-PAGE gel electrophoresis and protein concentration was protein and the C-terminal His-tag (Unzueta et al., 2012; determined by western blot using an anti-His monoclonal Serna et al., 2016). antibody (Genescript # A00186) with a calibration curve of ® Cultures of ClearColi BL21 (DE3) cells transformed with the plasmid pETDuet-IFN-γ-H6 were incubated in a shake flask at quantified soluble recombinant GFP-H6. 37°C and 250 rpm in LB medium supplemented with 100 μg/ml pETDuet-IFN-γ-H6 plasmid was transformed into ClearColi BL21 (DE3) cells and grown as described for the soluble protein ® ampicillin. Protein expression was induced in the same medium version. IB production was induced by adding 1 mmol/L by adding 1 mmol/L isopropyl-β-D-thiogalactopyranoside (IPTG). The cultures were then incubated at 20°C and isopropyl-β-D thiogalactopyranoside (IPTG) to ClearColi BL21 (DE3) cultures grown in LB supplemented with 100 μg/ ® 250 rpm O/N for protein production. Cells were harvested by ml ampicillin. After induction, the cultures were grown for 5 h at centrifugation (10 min at 5,000 g). IFN-γ-H6 was purified from 37°C. Cells were harvested by centrifugation (10 min at 5,000 g). the soluble cell fraction by affinity chromatography Purification of IBs of IFN-γ-H6 was performed following (Supplementary Method). The final protein concentration was protocol described in Supplementary Method. The determined by Bradford assay, yielding 0.80 mg/ml. recombinant protein yield was estimated by comparison with pNZ8148 plasmid (CmR, NICE) was transformed into a standard curve of known amounts of purified rBoIFN-γ protein competent L. lactis NZ9000 bacteria, as described elsewhere quantified by the Bradford assay. Quantification was performed (Cano-Garrido et al., 2016). Transformed L. lactis cells with with Image Lab software (Bio-Rad). pNZ8148-IFN-γ-H6 were grown in M17 medium pNZ8148-IFN-γ-H6 was transformed into L. lactis cells and supplemented with 0.5% glucose at 30°C without agitation. cells were grown as described in the production of soluble IFN-γ- Antibiotics were used for plasmid selection such as H6 protein version in this prokaryotic host. IB production was chloramphenicol (5 μg/ml) and erythromycin (2.5 μg/ml). induced by adding 500 ng/ml nisin (Sigma–Aldrich). After Protein production was induced in the same medium by induction, cultures were grown for 5 h. Final OD550 nm was Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 3 April 2022 | Volume 10 | Article 842256 Barguilla et al. Profiling Cytotoxicity of Inclusion Bodies 3.02. Bacteria were harvested at 5,000 g for 5min at 4°C and necrotic. Cells exposed for 30 min to 100 mM H2O2 were used washed twice with PBS. Purification of IBs of IFN-γ-H6 was as positive controls. performed following protocol described in Supplementary Method. Protein purity was determined by SDS-PAGE gel Comet Assay electrophoresis, and protein concentration was determined by The alkaline single cell gel electrophoresis (Comet) assay is a western blot using an anti-His monoclonal antibody (Genescript powerful technique to detect directly both oxidative stress and its # A00186) with a calibration curve of quantified soluble effects inducing DNA strand breaks at the level of individual cells. recombinant GFP-H6. Here it was used to evaluate the genotoxic and oxidative DNA Schematic representation of purification of IBs of T22-GFP- damage for caco-2 cells exposed to the different protein forms, using H6 and IFN-γ-H6 obtained from the insoluble cells fractions of formamidopyrimidine DNA glycosylase (FPG) as previously the expression hosts are showed in Supplementary Figure S1. described (Bach et al., 2014). Gelbond films (GF) were used for ® this assay. Cells were exposed for 24 h to high concentrations of the Cell Culture Conditions different recombinant proteins and forms: 4, 8, 18, 36, and 70 μg/ml. Caco-2 cells were grown in Dulbecco’s modified Eagle’s medium The next day, the cells were trypsinized, collected, centrifuged, and (DMEM) free of sodium pyruvate (Biowest, Nuaillé, France), resuspended in cold PBS 1X at a concentration of 1 × 106 cells/mL. In supplemented with 10% fetal bovine serum, 1% non-essential the following step, cells were mixed with 0.75% low-melting-point amino-acids (Biowest) and 2.5 μg/ml Plasmocin (InvivoGen, San agarose at 37°C and three drops of 7 μl each were placed in the GF. Diego, CA, United States) in a humidified atmosphere of 5% CO2 Two GF with identical samples were processed simultaneously in and 95% air at 37°C. This cell line was used as it provided low or absent each experiment. Then, a lysis step was performed overnight by expression of CXCR4 to which T22 peptide acted as an antagonist immersing the GF in ice-cold lysis buffer at 4°C (2.5 mol/L NaCl, (Tamamura et al., 2006; Rubie et al., 2011; Stuckel et al., 2020). 0.1 mol/L Na2EDTA, 0.1 mol/L Tris base, 1% Triton X-100, 1% lauroyl sarcosinate, 10% DMSO), at pH 10. The following day, the Cell Viability Assay GF replicates were gently washed twice (1 × 5 min, 1 × 50 min) in Cell viability was determined by the Beckman counter method enzyme buffer at 4°C and pH 8 (10 mmol/L HEPES, 0.1 mol/L KCl, using a ZTM Series Coulter-Counter (Beckman Coulter Inc. Bea, 0.5 mm l/L EDTA, 0.2 mg/ml BSA) and then, they were incubated CA, United States). Briefly, 150,000 cells per well were seeded on for 30 min at 37°C in enzyme buffer (negative control) or FPG- 12-well plates. The next day, the cells were exposed to increasing containing enzyme buffer. Later, the GF were washed with concentrations of the different protein formats of T22-GFP-H6 electrophoresis buffer and placed in a horizontal electrophoresis (ranging from 4 × 103 to 70 × 103 ng/mL, or what is the same, 0,14 tank for 35 min to allow the DNA unwinding 0.3 ml/L NaOH and to 2.3 μmol/L) and IFN-γ-H6 (ranging from 18 to 180 ng/ml, or 1 mmol/L Na2EDTA pH 13.2 before the electrophoresis, which was the same, 1–10 μmol/L). After 24 h, cells were counted, and carried out for 20 min at 0.8 V/cm and 300 mA at 4°C. Then, GF survival curves were represented. were rinsed with cold PBS for 15 min and fixed in absolute ethanol for 2 h before air-drying it overnight at room temperature. A staining Cell Death, Necrosis, and Apoptosis Assay step was performed by incubating the GF for 20 min with SYBR The proportion of live, apoptotic, and necrotic cells was assessed Gold 1/10,000 in TE buffer (10 mmol/L Tris, 1 mmol/L EDTA pH with Annexin V FLUOS staining kit (Sigma–Aldrich) following 7.5). Finally, gels were mounted, visualized for comets using an the manufacturer’s protocol. Thus, cells were seeded at a density epifluorescent microscope at ×20 magnification, and analyzed with of 150,000 cells per well in 12-well plates and the following day, the Komet 5.5 Image analysis system (Kinetic Imaging Ltd., they were treated with two different concentrations of each Liverpool, United Kingdom). The levels of DNA damage were protein: 18 × 103 ng/ml and 70 × 103 ng/ml T22-GFP-H6, and evaluated according to the percentage of DNA in the tail of the 72 and 180 ng/ml IFN-γ-H6. After 24 h of exposure, the cells. One hundred randomly selected comet images were analyzed supernatant was collected in a 15 ml tube. Adherent cells were per sample. Cells exposed to the DNA damaging agent methyl trypsinized and collected in the same tube as the supernatant for methanesulfonate (MMS,Sigma–Aldrich) at 200 μmol/L for 1 h at each condition. The mix was centrifuged and washed once with 37°C were used as controls. PSB 1X. The pellet was resuspended in 100 µl of incubation buffer with 2 µl of Annexin V and 2 µl of propidium iodide (PI). Statistics Unstained cells and resuspended cell mixture were used as Analysis of variance followed by Dunnett’s multiple comparison compensation controls to improve the gating strategy. The test was performed appropriately to compare the effect of the cells were incubated for 15 min at room temperature and in exposure to the different recombinant proteins. A two-sided p < the dark for staining before adding 100 µl more of incubation 0.05 was considered statistically significant in all cases. buffer per sample. Then, samples were analyzed by BD FACSCanto Flow Cytometer (BD Biosciences, Franklin Lakes, NJ, United States). More than 10,000 events per sample were RESULTS analyzed. Among these events, the PI-negative and Annexin V-negative cells represent the living population, PI-negative Analysis of Purified Recombinant Proteins and Annexin V-positive cells are considered apoptotic and Gel electrophoresis of T22-GFP-H6 and IFN-γ-H6 showed the cells positive for both PI and Annexin V are considered expected protein bands (Supplementary Figure S2). In the Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 4 April 2022 | Volume 10 | Article 842256 Barguilla et al. Profiling Cytotoxicity of Inclusion Bodies FIGURE 1 | Cytotoxicity of recombinant proteins. (A,C,E) Cell viability of caco-2 exposed for 24 h to increasing doses of INF-γ-H6 from ClearColi® , INF-γ-H6 from L. lactis and T22-GFP-H6, respectively. Data are presented as number of exposed cells relative to the non-exposed controls ±SEM. (B,D,F) Percentage of live, apoptotic, and necrotic cells in the non-exposed controls, and cells exposed for 24 h to increasing doses of INF-γ-H6 from ClearColi® , INF-γ-H6 from L. lactis and T22- GFP-H6, respectively. The positive control corresponds to the percentage of live, apoptotic, and necrotic cells after 30 min of exposure to H2O2. The data determined by Annexin V staining are presented as mean % ± SEM. soluble cell fraction (Supplementary Figure S2A) the main described (Céspedes et al., 2016). Protein identification in the protein band corresponded to the recombinant protein insoluble cell fraction was performed by immunodetection showing a purity >90%. Some other accompanying protein (Supplementary Figure S2B). In each sample, the protein bands of higher molecular weight corresponded to a small band compatible with the corresponding expected molecular proportion of quaternary structures. In fact, dimerization of weight could be detected although analysis on polyacrylamide GFP (Wiedenmann et al., 2000) and IFN-γ (Fensterl and Sen, gels showed variable purity (Supplementary Figure S3). In fact, 2009) has been widely described and observed even under the relative presence of the recombinant protein in such samples denaturing conditions of the SDS-PAGE electrophoresis. The depends on the protein itself and the experimental conditions set presence of protein NPs in the T22-GFP-H6 sample was during gene expression with a wide range of purity between determined by DLS (Supplementary Figure S4) as previously experiments for different proteins (Peternel et al., 2008; Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 5 April 2022 | Volume 10 | Article 842256 Barguilla et al. Profiling Cytotoxicity of Inclusion Bodies FIGURE 2 | Genotoxic and oxidative DNA damage induced by the recombinant proteins. DNA damage level on cells exposed to increasing doses of (A) INF-γ-H6 from ClearColi® , (B) INF-γ-H6 from L. lactis, and (C) T22-GFP-H6 after 24 h. 1 h exposure to MMS (200 μmol/L) was used as a positive control. Data are presented as mean % of DNA quantified in the comet tail ±SEM. Castellanos-Mendoza et al., 2014; Carratalá et al., 2020b). The 180 ng/ml for IFN-γ-H6; and 18 × 103 and 70 × 103 ng/mL of presence of associated proteins as chaperones and other cellular T22-GFP-H6), confirming the results of the cell viability assays components in IBs have been widely described (Ventura and (Figures 1B,D,F). Approximately 2% of the cell population Villaverde, 2006). were apoptotic cells in those exposed to IFN-γ-H6, while around 4% of the cells were apoptotic after exposure to T22-GFP-H6. Necrotic cells represented 1% of the caco-2 Toxicological Assessment of the Recombinant Proteins exposed to IFN-γ-H6 from L. Lactis, and 10% of the cells ® cells exposed to IFN-γ-H6 from ClearColi , 6% of the cells The analysis of diverse endpoints linked to the toxicity exposed to T22-GFP-H6. Nonetheless, none of these potential in vitro showed that neither T22-GFP-H6 nor percentages were significantly different from the ® IFN-γ-H6 purified from ClearColi or L. lactis were toxic in any of the protein forms assessed (natural IBs or soluble proportions observed in non-exposed cells. In line with the above, the analysis of oxidized DNA bases by the comet assay protein formats). Caco-2 cells had a cell survival rate with the inclusion of FPG enzyme also points out toward the generally over 80% when exposed to concentrations within absence of induced oxidative stress, as oxidative DNA damage the standard range, and no significant differences were found did not exceed 10% for any of the conditions tested (Figure 2). between exposed cells and non-exposed controls (Figures Importantly, the analysis of DNA damage in individual cells by 1A,C,E). This was further confirmed by the results from the the Comet assay revealed no genotoxic effects of the PI and Annexin V staining followed by flow cytometry recombinant proteins despite the increment in the dose of analysis, where living cells were again found to be above exposure. Genotoxic DNA damage levels did not differ from 80% for all concentrations tested (Figures 1B,D,F), and in those found in the control cells, with values no higher than any case, these percentages were significantly different from 12% (Figure 2). There were no significant differences in the the non-exposed controls. The proportion of apoptotic and levels of DNA damage when comparing T22-GFP-H6, IFN-γ- necrotic cells in the culture was characterized for an intermediate and a high concentration of exposure (72 and ® H6 from ClearColi , and IFN-γ-H6 from L. lactis, nor between IBs or the soluble form of the different proteins. Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 6 April 2022 | Volume 10 | Article 842256 Barguilla et al. Profiling Cytotoxicity of Inclusion Bodies DISCUSSION 2020a; Falgàs et al., 2020b; Céspedes et al., 2020; Serna et al., 2021). The IFN-γ proteins, in both formats, have been assayed in Recombinant proteins play an essential role in diagnosis and therapy. cultured cells and in animal models to check the The E. coli expression system is one of the most established hosts, immunostimulatory effect of the cytokine in infectious diseases generating more than 20% of biopharmaceuticals on the market (Carratalá et al., 2020a; Carratalá et al., 2020c). The lack of (Baeshen et al., 2014). However, although the steady increase in induction of cytotoxicity, cell death, apoptosis, and necrosis, mAbs approvals favors the trend toward mammalian over non- oxidative stress, and genotoxicity of any of the proteins and mammalian expression hosts (Walsh, 2018), in cancer therapies, the protein formats tested in this study indicates that this type of prokaryotic host-derived products account for more than 60% of protein complex may represent an appealing platform of protein- marketed products (Sanchez-Garcia et al., 2016). based biomaterials from which to explore their use in the One of the disadvantages of recombinant protein production development of novel protein formats in biomedical in E. coli is the formation of IBs, which have been classically applications including diagnosis and therapy. regarded as undesirable by-products. However, the detailed characterization of this type of protein aggregate has revealed the presence of protein molecules with native-like structure, DATA AVAILABILITY STATEMENT which can be recovered by diffusion from the scaffold structure of the IBs under non-denaturing conditions (Peternel The raw data that support the findings of this work are available et al., 2008; Singh et al., 2015; Gifre-Renom et al., 2018). In in UAB Digital Repository of Documents at https://ddd.uab.cat/ addition, IBs have been demonstrated to facilitate cell growth of record/257475. cultured cells on decorated surfaces and 3D scaffolds as well as to release recombinant protein in a sustained manner when administered subcutaneously (Seras-Franzoso et al., 2012; AUTHOR CONTRIBUTIONS Seras-Franzoso et al., 2013; Céspedes et al., 2020). These properties have raised interest in this biomaterial for AV, UU, NF-M, and AH contributed to the conception and biomedical applications (De Marco et al., 2019). For this design of the study. IB, UU, JC, and OC-G conducted the reason, toxicological assessment of IBs needs to be addressed. experiments and analyzed the data. IB and NF-M wrote the In this study, IBs were obtained from three different first draft of the manuscript. All authors contributed to the article prokaryotic expression hosts in order to discriminate the and read and approved the submitted version. putative toxicity of IBs obtained from E. coli, which produces ® endotoxins; from an E. coli strain (ClearColi ) that produces a genetically modified version of lipopolysaccharide (LPS), which FUNDING does not induce endotoxin response in mammalian cells; and from L. lactis, an LPS-free host (Wakelin et al., 2006; Morello NF-M received support from AIE (PID2019-107298RB-C22). AH et al., 2008; Mamat et al., 2015). In all cases, two protein formats received support from AIE (PID2020-116789, RB-C43). UU was were obtained, the soluble protein and the corresponding IBs, for supported by a Miguel Servet contract (CP19/ 00028) from ISCIII comparison reasons. The results presented here indicate that co-funded by the European Social Fund (ESF investing in your neither the protein format nor the origin of the protein samples future) and ISCIII (PI20/00400) co-funded by the European had any effect on the toxicity assays performance on caco-2 cells. Regional Development Fund (ERDF, a way to make Europe). The presence of endotoxins in the protein samples obtained from AV was granted with an ICREA ACADEMIA award. E. coli has been widely described for soluble counterparts (Schwarz et al., 2014) and in IB products (Stanek et al., 2019). Even though the LPS contents of the protein samples obtained in ACKNOWLEDGMENTS this study were not determined, it can be assumed that this product was present at the same level. In the case of proteins Protein production was partially performed by the ICTS obtained from the other two expression systems, the presence of ‘‘NANBIOSIS,” more specifically by the Protein Production LPS might be residual and only a product of cross-contamination Platform of CIBER in Bioengineering, Biomaterials, and arising from sharing the same laboratory facilities, equipment, Nanomedicine (CIBER-BBN)/IBB, at the UAB http://www. and material. In fact, the protein concentrations used in this study nanbiosis.es/portfolio/u1-protein-production-platform-ppp/. mirrored previous experimental approaches used in cell culture The authors appreciate the technical support of Rosa Mendoza assays to assess the biological activity of the recombinant proteins and Mercedes Márquez. (Serna et al., 2019; Carratalá et al., 2020a; Carratalá et al., 2020c; Álamo et al., 2020). The soluble version and the IBs format of T22-GFP-H6 have been tested for the selective interaction with SUPPLEMENTARY MATERIAL CXCR4+ cells to target metastatic cells in a variety of cancer types as well as their performance as an antimicrobial agent due to the The Supplementary Material for this article can be found online at: presence of the T22 peptide (polyphemusin) isolated from the https://www.frontiersin.org/articles/10.3389/fbioe.2022.842256/ hemocytes of horseshoe crabs (Céspedes et al., 2016; Falgàs et al., full#supplementary-material Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 7 April 2022 | Volume 10 | Article 842256 Barguilla et al. Profiling Cytotoxicity of Inclusion Bodies REFERENCES Falgàs, A., Pallarès, V., Unzueta, U., Céspedes, M. V., Arroyo-Solera, I., Moreno, M. J., et al. (2020b). A CXCR4-Targeted Nanocarrier Achieves Highly Selective Tumor Uptake in Diffuse Large B-Cell Lymphoma Mouse Models. Álamo, P., Pallarès, V., Céspedes, M. V., Falgàs, A., Sanchez, J. M., Serna, N., et al. Haematologica 105, 741–753. doi:10.3324/haematol.2018.211490 (2020). Fluorescent Dye Labeling Changes the Biodistribution of Tumor- Fensterl, V., and Sen, G. C. (2009). Interferons and Viral Infections. Biofactors 35, Targeted Nanoparticles. Pharmaceutics 12, 1004. doi:10.3390/ 14–20. doi:10.1002/biof.6 pharmaceutics12111004 Fenton, O. S., Olafson, K. N., Pillai, P. S., Mitchell, M. J., and Langer, R. (2018). Bach, J., Sampayo-Reyes, A., Marcos, R., and Hernández, A. (2014). Ogg1 Genetic Advances in Biomaterials for Drug Delivery. Adv. Mater. 30, 1705328. doi:10. Background Determines the Genotoxic Potential of Environmentally Relevant 1002/adma.201705328 Arsenic Exposures. Arch. Toxicol. 88, 585–596. doi:10.1007/s00204-013-1151-0 Ferrer-Miralles, N., Domingo-Espín, J., Corchero, J. L., Vázquez, E., and Villaverde, Baeshen, N. A., Baeshen, M. N., Sheikh, A., Bora, R. S., Ahmed, M. M. M., A. (2009). Microbial Factories for Recombinant Pharmaceuticals. Microb. Cel Ramadan, H. A. I., et al. (2014). Cell Factories for Insulin Production. Microb. Fact. 8, 17. doi:10.1186/1475-2859-8-17 Cel Fact. 13, 141. doi:10.1186/s12934-014-0141-0 Gifre-Renom, L., Cano-Garrido, O., Fàbregas, F., Roca-Pinilla, R., Seras-Franzoso, Baeshen, M. N., Al-Hejin, A. M., Bora, R. S., Ahmed, M. M. M., Ramadan, H. A. I., J., Ferrer-Miralles, N., et al. (2018). A New Approach to Obtain Pure and Active Saini, K. S., et al. (2015). Production of Biopharmaceuticals in E. coli: Current Proteins from Lactococcus Lactis Protein Aggregates. Sci. Rep. 8, 13917. doi:10. Scenario and Future Perspectives. J. Microbiol. Biotechnol. 25, 953–962. doi:10. 1038/s41598-018-32213-8 4014/jmb.1412.12079 Ibraheem, D., Elaissari, A., and Fessi, H. (2014). Administration Strategies for Cano-Garrido, O., Sánchez-Chardi, A., Parés, S., Giró, I., Tatkiewicz, W. I., Ferrer- Proteins and Peptides. Int. J. Pharmaceutics 477, 578–589. doi:10.1016/j. Miralles, N., et al. (2016). Functional Protein-Based Nanomaterial Produced in ijpharm.2014.10.059 Microorganisms Recognized as Safe: A New Platform for Biotechnology. Acta Jäger, V. D., Lamm, R., Küsters, K., Ölçücü, G., Oldiges, M., Jaeger, K.-E., et al. Biomater. 43, 230–239. doi:10.1016/j.actbio.2016.07.038 (2020). Catalytically-active Inclusion Bodies for Biotechnology-General Carratalá, J. V., Cano-garrido, O., Sánchez, J., Membrado, C., Pérez, E., Conchillo- Concepts, Optimization, and Application. Appl. Microbiol. Biotechnol. 104, Solé, O., et al. (2020a). Aggregation-prone Peptides Modulate Activity of Bovine 7313–7329. doi:10.1007/s00253-020-10760-3 Interferon Gamma Released from Naturally Occurring Protein Nanoparticles. Kopito, R. R. (2000). Aggresomes, Inclusion Bodies and Protein Aggregation. New Biotechnol. 57, 11–19. doi:10.1016/j.nbt.2020.02.001 Trends Cel Biol. 10, 524–530. doi:10.1016/s0962-8924(00)01852-3 Carratalá, J. V., Serna, N., Villaverde, A., Vázquez, E., and Ferrer-Miralles, N. López-Laguna, H., Unzueta, U., Conchillo-Solé, O., Sánchez-Chardi, A., (2020b). Nanostructured Antimicrobial Peptides: The Last Push towards Pesarrodona, M., Cano-Garrido, O., et al. (2019). Assembly of Histidine- Clinics. Biotechnol. Adv. 44, 107603. doi:10.1016/j.biotechadv.2020.107603 Rich Protein Materials Controlled through Divalent Cations. Acta Biomater. Carratalá, J. V., Brouillette, E., Serna, N., Sánchez-Chardi, A., Sánchez, J. M., 83, 257–264. doi:10.1016/j.actbio.2018.10.030 Villaverde, A., et al. (2020c). In Vivo Bactericidal Efficacy of GWH1 Magalhães, P. O., Lopes, A. M., Mazzola, P. G., Rangel-Yagui, C., Penna, T. C., and Antimicrobial Peptide Displayed on Protein Nanoparticles, a Potential Pessoa, A. (2007). Methods of Endotoxin Removal from Biological Alternative to Antibiotics. Pharmaceutics 12, 1217. doi:10.3390/ Preparations: a Review. J. Pharm. Pharm. Sci. 10, 388–404. pharmaceutics12121217 Mamat, U., Wilke, K., Bramhill, D., Schromm, A. B., Lindner, B., Kohl, T. A., et al. Carratalá, J. V., Gifre-Renom, L., Roca-Pinilla, R., Villaverde, A., Arís, A., Garcia- (2015). Detoxifying Escherichia coli for Endotoxin-free Production of Fruitós, E., et al. (2021). Selecting Subpopulations of High-Quality Protein Recombinant Proteins. Microb. Cel Fact. 14, 57. doi:10.1186/s12934-015- Conformers Among Conformational Mixtures of Recombinant Bovine MMP-9 0241-5 Solubilized from Inclusion Bodies. Int. J. Mol. Sci. 22, 3020. doi:10.3390/ Morello, E., Bermúdez-Humarán, L. G., Llull, D., Solé, V., Miraglio, N., Langella, P., ijms22063020 et al. (2008). Lactococcus Lactis, an Efficient Cell Factory for Recombinant Castellanos-Mendoza, A., Castro-Acosta, R. M., Olvera, A., Zavala, G., Mendoza- Protein Production and Secretion. J. Mol. Microbiol. Biotechnol. 14, 48–58. Vera, M., García-Hernández, E., et al. (2014). Influence of pH Control in the doi:10.1159/000106082 Formation of Inclusion Bodies during Production of Recombinant Murakami, T., Nakajima, T., Koyanagi, Y., Tachibana, K., Fujii, N., Tamamura, H., Sphingomyelinase-D in Escherichia coli. Microb. Cel Fact 13, 137. doi:10. et al. (1997). A Small Molecule CXCR4 Inhibitor that Blocks T Cell Line-Tropic 1186/s12934-014-0137-9 HIV-1 Infection. J. Exp. Med. 186, 1389–1393. doi:10.1084/jem.186.8.1389 Céspedes, M. V., Unzueta, U., Álamo, P., Gallardo, A., Sala, R., Casanova, I., et al. Pesarrodona, M., Jauset, T., Díaz-Riascos, Z. V., Sánchez-Chardi, A., Beaulieu, M. (2016). Cancer-specific Uptake of a Liganded Protein Nanocarrier Targeting E., Seras-Franzoso, J., et al. (2019). Targeting Antitumoral Proteins to Breast Aggressive CXCR4 + Colorectal Cancer Models. Nanomedicine: Nanotechnol. Cancer by Local Administration of Functional Inclusion Bodies. Adv. Sci. 6, Biol. Med. 12, 1987–1996. doi:10.1016/j.nano.2016.04.003 1900849. doi:10.1002/advs.201900849 Céspedes, M. V., Cano-Garrido, O., Álamo, P., Sala, R., Gallardo, A., Serna, N., et al. Peternel, Š., Grdadolnik, J., Gaberc-Porekar, V., and Komel, R. (2008). Engineering (2020). Engineering Secretory Amyloids for Remote and Highly Selective Destruction Inclusion Bodies for Non Denaturing Extraction of Functional Proteins. of Metastatic Foci. Adv. Mater. 32, 1907348. doi:10.1002/adma.201907348 Microb. Cel Fact 7, 34. doi:10.1186/1475-2859-7-34 Cheng, X., Tian, X., Wu, A., Li, J., Tian, J., Chong, Y., et al. (2015). Protein Corona Petitcollin, A., Bensalem, A., Verdier, M.-C., Tron, C., Lemaitre, F., Paintaud, G., Influences Cellular Uptake of Gold Nanoparticles by Phagocytic and et al. (2020). Modelling of the Time-Varying Pharmacokinetics of Therapeutic Nonphagocytic Cells in a Size-dependent Manner. ACS Appl. Mater. Inter. Monoclonal Antibodies: A Literature Review. Clin. Pharmacokinet. 59, 37–49. 7, 20568–20575. doi:10.1021/acsami.5b04290 doi:10.1007/s40262-019-00816-7 De Marco, A., Ferrer-Miralles, N., Garcia-Fruitós, E., Mitraki, A., Peternel, S., Planesse, C., Nativel, B., Iwema, T., Gasque, P., Robert-Da Silva, C., and Rinas, U., et al. (2019). Bacterial Inclusion Bodies Are Industrially Exploitable Viranaïcken, W. (2015). Recombinant Human HSP60 Produced in Amyloids. FEMS Microbiol. Rev. 43, 53–72. doi:10.1093/femsre/fuy038 ClearColi BL21(DE3) Does Not Activate the NFκB Pathway. Cytokine 73, Díez-Gil, C., Krabbenborg, S., García-Fruitós, E., Vazquez, E., Rodríguez- 190–195. doi:10.1016/j.cyto.2015.01.028 Carmona, E., Ratera, I., et al. (2010). The Nanoscale Properties of Bacterial Puertas-Bartolomé, M., Mora-Boza, A., and García-Fernández, L. (2021). Inclusion Bodies and Their Effect on Mammalian Cell Proliferation. Emerging Biofabrication Techniques: A Review on Natural Polymers for Biomaterials 31, 5805–5812. doi:10.1016/j.biomaterials.2010.04.008 Biomedical Applications. Polymers 13, 1209. doi:10.3390/polym13081209 Ecker, D. M., Jones, S. D., and Levine, H. L. (2015). The Therapeutic Monoclonal Rodríguez-Carmona, E., Mendoza, R., Ruiz-Cánovas, E., Ferrer-Miralles, N., Antibody Market. MAbs 7, 9–14. doi:10.4161/19420862.2015.989042 Abasolo, I., Schwartz, S., et al. (2015). A Novel Bio-Functional Material Falgàs, A., Pallarès, V., Serna, N., Sánchez-García, L., Sierra, J., Gallardo, A., et al. Based on Mammalian Cell Aggresomes. Appl. Microbiol. Biotechnol. 99, (2020a). Selective Delivery of T22-PE24-H6 to CXCR4+ Diffuse Large B-Cell 7079–7088. doi:10.1007/s00253-015-6684-0 Lymphoma Cells Leads to Wide Therapeutic index in a Disseminated Mouse Rubie, C., Frick, V. O., Ghadjar, P., Wagner, M., Justinger, C., Faust, S. K., et al. Model. Theranostics 10, 5169–5180. doi:10.7150/thno.43231 (2011). CXC Receptor-4 mRNA Silencing Abrogates CXCL12-Induced Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 8 April 2022 | Volume 10 | Article 842256 Barguilla et al. Profiling Cytotoxicity of Inclusion Bodies Migration of Colorectal Cancer Cells. J. Transl. Med. 9, 22. doi:10.1186/1479- Science-Present and Future. Editor Y. Shimonishi (Dordrecht: Springer 5876-9-22 Netherlands), 398–402. doi:10.1007/0-306-46864-6_131 Rueda, F., Gasser, B., Sánchez-Chardi, A., Roldán, M., Villegas, S., Puxbaum, V., Tatkiewicz, W. I., Seras-Franzoso, J., García-Fruitós, E., Vazquez, E., Kyvik, A. R., et al. (2016). Functional Inclusion Bodies Produced in the Yeast Pichia pastoris. Ventosa, N., et al. (2019). High-Throughput Cell Motility Studies on Surface- Microb. Cel Fact. 15, 166. doi:10.1186/s12934-016-0565-9 Bound Protein Nanoparticles with Diverse Structural and Compositional Sanchez-Garcia, L., Martín, L., Mangues, R., Ferrer-Miralles, N., Vázquez, E., and Characteristics. ACS Biomater. Sci. Eng. 5, 5470–5480. doi:10.1021/ Villaverde, A. (2016). Recombinant Pharmaceuticals from Microbial Cells: A acsbiomaterials.9b01085 2015 Update. Microb. Cel Fact. 15, 33. doi:10.1186/s12934-016-0437-3 Unzueta, U., Ferrer-Miralles, N., Cedano, J., Zikung, X., Pesarrodona, M., Saccardo, Schroder, K., Hertzog, P. J., Ravasi, T., and Hume, D. A. (2004). Interferon-γ: an P., et al. (2012). Non-amyloidogenic Peptide Tags for the Regulatable Self- Overview of Signals, Mechanisms and Functions. J. Leukoc. Biol. 75, 163–189. Assembling of Protein-Only Nanoparticles. Biomaterials 33, 8714–8722. doi:10. doi:10.1189/jlb.0603252 1016/j.biomaterials.2012.08.033 Schwarz, H., Schmittner, M., Duschl, A., and Horejs-Hoeck, J. (2014). Residual Unzueta, U., Serna, N., Sánchez-García, L., Roldán, M., Sánchez-Chardi, A., Endotoxin Contaminations in Recombinant Proteins Are Sufficient to Activate Mangues, R., et al. (2017). Engineering Multifunctional Protein Human CD1c+ Dendritic Cells. PLoS One 9, e113840. doi:10.1371/journal. Nanoparticles by In Vitro Disassembling and Reassembling of Heterologous pone.0113840 Building Blocks. Nanotechnology 28, 505102. doi:10.1088/1361-6528/aa963e Seras-Franzoso, J., Díez-Gil, C., Vazquez, E., García-Fruitós, E., Cubarsi, R., Ratera, Ventura, S., and Villaverde, A. (2006). Protein Quality in Bacterial Inclusion I., et al. (2012). Bioadhesiveness and Efficient Mechanotransduction Stimuli Bodies. Trends Biotechnol. 24, 179–185. doi:10.1016/j.tibtech.2006.02.007 Synergistically provided by Bacterial Inclusion Bodies as Scaffolds for Tissue Viranaicken, W., Nativel, B., Krejbich-Trotot, P., Harrabi, W., Bos, S., El Engineering. Nanomedicine 7, 79–93. doi:10.2217/nnm.11.83 Kalamouni, C., et al. (2017). ClearColi BL21(DE3)-based Expression of Zika Seras-Franzoso, J., Steurer, C., Roldán, M., Vendrell, M., Vidaurre-Agut, C., Virus Antigens Illustrates a Rapid Method of Antibody Production against Tarruella, A., et al. (2013). Functionalization of 3D Scaffolds with Protein- Emerging Pathogens. Biochimie 142, 179–182. doi:10.1016/j.biochi.2017.09.011 Releasing Biomaterials for Intracellular Delivery. J. Control. Release 171, 63–72. Wakelin, S. J., Sabroe, I., Gregory, C. D., Poxton, I. R., Forsythe, J. L. R., Garden, O. doi:10.1016/j.jconrel.2013.06.034 J., et al. (2006). "Dirty Little Secrets"-Endotoxin Contamination of Recombinant Serna, N., Céspedes, M. V., Saccardo, P., Xu, Z., Unzueta, U., Álamo, P., et al. Proteins. Immunol. Lett. 106, 1–7. doi:10.1016/j.imlet.2006.04.007 (2016). Rational Engineering of Single-Chain Polypeptides into Protein-Only, Walsh, G. (2018). Biopharmaceutical Benchmarks 2018. Nat. Biotechnol. 36, BBB-Targeted Nanoparticles. Nanomedicine: Nanotechnol. Biol. Med. 12, 1136–1145. doi:10.1038/nbt.4305 1241–1251. doi:10.1016/j.nano.2016.01.004 Wiedenmann, J., Elke, C., Spindler, K.-D., and Funke, W. (2000). Cracks in the β- Serna, N., Sánchez, J. M., Unzueta, U., Sánchez-García, L., Sánchez-Chardi, A., can: Fluorescent Proteins from Anemonia Sulcata (Anthozoa, Actinaria). Proc. Mangues, R., et al. (2019). Recruiting Potent Membrane Penetrability in Tumor Natl. Acad. Sci. U.S.A. 97, 14091–14096. doi:10.1073/pnas.97.26.14091 Cell-Targeted Protein-Only Nanoparticles. Nanotechnology 30, 115101. doi:10. Xu, Y., Yasin, A., Tang, R., Scharer, J. M., Moo-Young, M., and Chou, C. P. (2008). 1088/1361-6528/aaf959 Heterologous Expression of Lipase in Escherichia coli Is Limited by Folding and Serna, N., Carratalá, J. V., Conchillo-Solé, O., Martínez-Torró, C., Unzueta, U., Disulfide Bond Formation. Appl. Microbiol. Biotechnol. 81, 79–87. doi:10.1007/ Mangues, R., et al. (2021). Antibacterial Activity of T22, a Specific Peptidic s00253-008-1644-6 Ligand of the Tumoral Marker CXCR4. Pharmaceutics 13, 1922. doi:10.3390/ pharmaceutics13111922 Conflict of Interest: The authors declare that the research was conducted in the Singh, A., Upadhyay, V., Upadhyay, A. K., Singh, S. M., and Panda, A. K. (2015). absence of any commercial or financial relationships that could be construed as a Protein Recovery from Inclusion Bodies of Escherichia coli Using Mild potential conflict of interest. Solubilization Process. Microb. Cel Fact. 14, 41. doi:10.1186/s12934-015- 0222-8 Publisher’s Note: All claims expressed in this article are solely those of the authors Stanek, O., Masin, J., Osicka, R., Jurnecka, D., Osickova, A., and Sebo, P. (2019). and do not necessarily represent those of their affiliated organizations, or those of Rapid Purification of Endotoxin-free RTX Toxins. Toxins 11, 336. doi:10.3390/ the publisher, the editors, and the reviewers. Any product that may be evaluated in toxins11060336 this article, or claim that may be made by its manufacturer, is not guaranteed or Stuckel, A. J., Zhang, W., Zhang, X., Zeng, S., Dougherty, U., Mustafi, R., et al. endorsed by the publisher. (2020). Enhanced CXCR4 Expression Associates with Increased Gene Body 5- Hydroxymethylcytosine Modification but Not Decreased Promoter Copyright © 2022 Barguilla, Unzueta, Carratalá, Cano-Garrido, Villaverde, Methylation in Colorectal Cancer. Cancers 12, 539. doi:10.3390/ Hernández and Ferrer-Miralles. This is an open-access article distributed under CANCERS12030539 the terms of the Creative Commons Attribution License (CC BY). The use, Tamamura, H., Matsumoto, F., Sakano, K., Otaka, A., Murakami, T., Nakashima, distribution or reproduction in other forums is permitted, provided the original H., et al. (2006). “An Anti-HIV Peptide T22 Is Confirmed to Be a Selective author(s) and the copyright owner(s) are credited and that the original publication CXCR4/fusin Inhibitor by Comparative Inhibition Study of a Chemokine in this journal is cited, in accordance with accepted academic practice. No use, SDF-1, Synthesized by Regioselective Disulfide Bond Formation,” in Peptide distribution or reproduction is permitted which does not comply with these terms. Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 9 April 2022 | Volume 10 | Article 842256