Structural and functional features of self-assembling protein nanoparticles produced in endotoxin-free Escherichia coli
- Fabián Rueda1, 2, 3,
- María Virtudes Céspedes3, 4,
- Alejandro Sánchez-Chardi5,
- Joaquin Seras-Franzoso1, 2, 3, 9,
- Mireia Pesarrodona1, 2, 3,
- Neus Ferrer-Miralles1, 2, 3,
- Esther Vázquez1, 2, 3,
- Ursula Rinas6, 7,
- Ugutz Unzueta3, 4,
- Uwe Mamat8,
- Ramón Mangues3, 4,
- Elena García-Fruitós1, 2, 3, 10 and
- Antonio Villaverde1, 2, 3Email author
© Rueda et al. 2016
Received: 21 January 2016
Accepted: 28 March 2016
Published: 8 April 2016
Production of recombinant drugs in process-friendly endotoxin-free bacterial factories targets to a lessened complexity of the purification process combined with minimized biological hazards during product application. The development of nanostructured recombinant materials in innovative nanomedical activities expands such a need beyond plain functional polypeptides to complex protein assemblies. While Escherichia coli has been recently modified for the production of endotoxin-free proteins, no data has been so far recorded regarding how the system performs in the fabrication of smart nanostructured materials.
We have here explored the nanoarchitecture and in vitro and in vivo functionalities of CXCR4-targeted, self-assembling protein nanoparticles intended for intracellular delivery of drugs and imaging agents in colorectal cancer. Interestingly, endotoxin-free materials exhibit a distinguishable architecture and altered size and target cell penetrability than counterparts produced in conventional E. coli strains. These variant nanoparticles show an eventual proper biodistribution and highly specific and exclusive accumulation in tumor upon administration in colorectal cancer mice models, indicating a convenient display and function of the tumor homing peptides and high particle stability under physiological conditions.
The observations made here support the emerging endotoxin-free E. coli system as a robust protein material producer but are also indicative of a particular conformational status and organization of either building blocks or oligomers. This appears to be promoted by multifactorial stress-inducing conditions upon engineering of the E. coli cell envelope, which impacts on the protein quality control of the cell factory.
The physiological diversity of microorganisms makes them suitable platforms for the cost-effective fabrication of a diversity of substances and macromolecules, including emerging nanostructured materials such as metal deposits, polymeric granules, functional amyloids and viruses or virus-like protein assemblies . Being highly versatile, environmentally friendly and fully scalable, microbial fabrication is in many aspects more convenient than chemical synthesis [2–4]. Microbial cells, namely bacteria and yeast, have been exploited as factories for the production of many protein drugs that have been approved for use in humans. Historically, Escherichia coli was the pioneering cell factory for protein production because of the well-known genetics and metabolism. However, the presence of endotoxins in the cell envelope of Gram-negative bacteria poses an up to now unsolvable obstacle in the use of this organism for drug production . Lipopolysaccharide (LPS) removal increases the complexity of protein purification processes, and it is recognized that LPS, even in low amounts, is a common contaminant of complex protein complexes such as bacteriophages  and also of plain recombinant proteins . In fact, contaminant LPS and other cell envelope components are responsible for wrongly attributed biological effects of recombinant proteins tested in biomedical assays, and for poor biocompatibility (some examples can be found in [8–10]). In this context, Gram-positive bacteria, yeast and mammalian cells are endotoxin-free alternatives to E. coli, and their prevalence in protein drug production has steadily increased [11, 12], although not absent of problems.
Very recently , endotoxin-free E. coli strains have been developed by fine multi-step mutagenesis as a way to merge the versatility of E. coli as an advantageous protein production platform in the absence of endotoxic contaminants in its products. Those strains have been proved efficient in the production of diverse soluble protein species  and also of inclusion bodies intended as smart topologies  or as functional amyloids for intracellular protein release . We wanted here to explore how the production, functionalities and nanoarchitecture of a complex, virus-like protein nanoparticle (T22-GFP-H6) designed as drug carrier  would be affected by the use of this particular fabrication platform. These particles are formed by a self-organizing, CXCR4-targeted polypeptide that accumulates, in the assembled form, in tumoral tissues of colorectal cancer animal models [16, 17]. Accumulation in tumor is promoted by the N-terminal peptide T22, which is a potent ligand of the cell surface cytokine receptor CXCR4 . CXCR4 is overexpressed in colorectal cancer cells and linked to tumor aggressiveness . The combination of the highly cationic T22 peptide with the C-terminal histidine tail in a modular protein promotes the self-assembly of the whole construct, favored by the bipolar charge distribution of the building block surface . Recently, we have observed that strain-dependent genetic features of the producing bacteria impact on tumor targeting and on the whole-body biodistribution of the oligomeric construct upon its systemic administration . Then, we were interested in dissecting the structure and activities of these tumor-homing nanoparticles when produced in endotoxin-free E. coli cells, whose complex genetic development might have resulted in relevant physiological shifts.
Strains, plasmids and media
T22-GFP-H6 is a tumor-targeted modular protein that self-assembles as toroid nanoparticles of about 12 nm of diameter. These oligomers are fully stable in vivo upon tail vein administration in colorectal cancer mice models  and accumulate intracellularly in primary tumor and in metastatic foci . T22-GFP-H6 protein nanoparticles were produced in the KPM22 L11-derivative , endotoxin-free strain KPM335 (msbA52, ΔgutQ, ΔkdsD, ΔlpxL, ΔlpxM, ΔpagP, ΔlpxP, ΔeptA, frr181) and its parental K-12 strain BW30270 (CGSC#7925–MG1655; F−, rph +, fnr +) with a wild-type LPS. The design of KPM335, based on the incorporation of non-reverting deletions of seven genes to disrupt Kdo biosynthesis and modifications of lipid IVA, prevents the strain from regaining the potential to synthesize normal LPS or endotoxically active lipid IVA derivatives through acquiring mutations. Also, E. coli K-12 MC4100 ([araD139], (argF-lac)169, λ—relA1, rpsL150, rbsR22, flb5301, deoC1, pstF25 StrepR) and BL21 Origami B [F− ompT hsdS B (r B − m B − ) gal dcm lacY1 ahpC (DE3) gor522::Tn10 trxB (KanR, TetR)] (Novagen, Darmstadt, Germany) were used for protein production as controls. KPM335, BW30270 and MC4100 were transformed with plasmid pTrc99a, whereas Origami B was transformed with a pET22b-derived plasmid. All expression plasmids contained the T22-GFP-H6 DNA sequence optimized by the codon usage of E. coli and have been described previously . Bacteria were always grown in Lysogeny Broth (LB) rich media . Part of the protein was produced with the assistance of the Protein Production Platform of CIBER-BBN/IBB, at the UAB (http://www.nanbiosis.es/unit/u1-protein-production-platform-ppp/).
Competent cells and transformation
Cultures were grown overnight at 37 °C with shaking at 250 rpm and used as a 1/100 inoculum in 50 ml of LB. After reaching an optical density (OD550) between 0.2 and 0.4, MC4100, BW30270 and Origami B cultures were centrifuged (4000×g) at 4 °C for 15 min. Pellets were resuspended in 12.5 ml of cold and sterile 50 mM CaCl2 and incubated for 45 min in an ice bath. Cells were centrifuged again as described above and resuspended in 1.25 ml of cold and sterile 50 mM CaCl2 in glycerol (15 % v/v) to prepare aliquots of 200 µl and stored at −80 °C. To transform the cells, 40 ng of plasmid DNA were added to the competent cells. The mixtures were incubated on ice for 30–60 min, warmed up to 42 °C for 45 s and placed on ice for 30 s. After incubation, 800 µl of LB media were added, and transformed cells were incubated at 37 °C for 1 h. Finally, the cells were plated on LB-agar plates containing the corresponding antibiotic. In the case of KMP335 strain, cells were grown to an OD550 between 0.2 and 0.4, placed on ice for 20 min and sedimented by centrifugation (3100×g, 4 °C, 20 min). The pellets were centrifuged and resuspended successively in 40, 20 and 10 ml of H2O (ice-cold and sterile), followed by a wash with 5 ml of 10 % glycerol (ice-cold and sterile). The final pellet was resuspended in 1 ml of 10 % glycerol (ice-cold and sterile) to prepare 50-µl aliquots for storage at −80 °C. Electrocompetent KPM335 cells were transformed by electroporation in pre-chilled 0.2 cm electroporation cuvettes using 50 µl of competent cells and 40 ng of plasmid DNA. Cells were pulsed using a Gene Pulser MX cell electroporator (Bio-Rad, Hercules, CA, USA) at 25 µF, 200 Ω, 2500 V and 4.7–4.8 ms. Immediately after the pulse, 800 µl of LB medium were added, and the mixture was incubated at 37 °C for 1 h. Cells were plated on LB-agar plates containing ampicillin (100 µg/ml) and incubated at 37 °C overnight.
Protein production and purification
Overnight cultures were inoculated in 500 ml of LB media with 100 µg/ml ampicillin in 2-L flasks. Streptomycin (at 30 µg/ml) was also added to MC4100 cultures, and tetracycline (100 µg/ml) and kanamycin (33 µg/ml) to Origami B cultures. Each culture was grown at 37 °C and 250 rpm to an OD550 of about 0.5 before isopropyl-β-d-thiogalactoside (IPTG) was added to 1 mM to induce recombinant gene expression. T22-GFP-H6 was produced overnight at 20 °C with shaking (250 rpm) in all cases.
After overnight production, cultures were centrifuged (3280×g, 4 °C, 40 min), and the cell pellets were resuspended in 20 mM Tris–HCl, pH 8.0, containing 500 mM NaCl, 20 mM imidazole (buffer A), and an EDTA-free protease inhibitor cocktail (Complete EDTA-free, Roche Diagnostics, Indianapolis, IN, USA). Intra-cellular protein was extracted by cell disruption using a French press (Thermo FA-078A) at 1100 psi, and purified by His-tag affinity chromatography using a 1-ml HiTrap Chelating HP column (GE Healthcare, Piscataway, NJ, USA) in an ÄKTA purifier FPLC (GE Healthcare). Protein separation was achieved with a linear imidazole gradient from 20 mM to 500 mM, by diluting 20 mM Tris–HCl, pH 8.0, plus 500 mM NaCl and 500 mM imidazole (buffer B) in buffer A, and fractions were collected and dialyzed against 166 mM NaHCO3, pH 7.4. The amount of protein was determined by Bradford’s assay , and the integrity of the protein was analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by Western blot analysis as described below.
Western blot analysis
Separated fractions were subjected to 10 % SDS-PAGE according to Laemmli’s method . Protein was transferred to nitrocellulose membranes (GE Healthcare, Piscataway, NJ, USA) that were blocked overnight with 5 % skim milk in PBS. After blocking, membranes were incubated with anti-GFP antibody (1:500, sc-8334, Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by incubation with a secondary HRP-conjugated anti-rabbit IgG (H + L) antibody (Bio-Rad, Hercules, CA, USA) at a dilution of 1:2000. Protein bands were visualized with a solution of 25 % cold methanol, 0.2 % H2O2 and 0.65 mg/ml of 4-chloronaftol in PBS, and images were obtained using a GS800 Calibrated Densitometer scanner (Bio-Rad).
Fluorescence intensity of T22-GFP-H6 was determined in a Varian Cary Eclipse fluorescence spectrometer (Agilent Technologies, Mulgrave, Australia) at excitation and emission wavelengths of 450 and 510 nm, respectively.
Particle size and zeta potential
Size distribution and zeta potential of nanoparticles were measured by dynamic light scattering (DLS) at 633 nm (Zetasizer Nano ZS, Malvern Instruments Limited, Malvern, Worcestershire, UK). Samples were analyzed by triplicate averaging of fifteen single measurements.
Mass spectrometry (MS) analysis
Purified protein was diluted 1:2 with H2O MilliQ and dialyzed against 50 mM of NH4HCO3 for 1.5 h. After dialysis, protein was mixed with a matrix of 2.6 dihydroxyacetophenone (1:1), and 1 µl of the mixture was deposited on a ground steel plate. Samples were analyzed using a lineal method in an UltrafleXtreme MALDI-TOF instrument (Bruker Daltonics, Bremen, Germany) with ion acceleration of 25 kV.
Circular dichroism (CD)
CD measurements were carried out in a Jasco J-715 spectropolarimeter at 25 °C and 10 µM protein in 160 mM NaHCO3, pH 7.4. CD and high-tension (HT) voltage spectra were obtained over a wavelength range of 200–250 nm at a scan rate of 50 nm/min, a response time of 0.5 s, and a bandwidth of 0.5 nm.
To visualize the ultrastructure of protein nanoparticles by transmission electron microscopy (TEM), 2 µl of purified protein (0.2 mg/ml) were placed on carbon-coated copper grids for 1 min. Excess of sample was bloated and 2 µl of 1 % w/v uranyl acetate were added for negative staining. Samples were immediately visualized in a TEM Jeol JEM-1400 (Jeol Ltd., Tokyo, Japan) equipped with a Gatan CCD Erlangshen ES1000 W camera (Gatan Inc, Abingdon, UK) and operating at 80 kV. Ultrastructural analyses of nanoparticle morphology were complemented with imaging in a nearly native state with a Field Emission Scanning Electron Microscope (FESEM). For that, protein samples were directly deposited over silicon wafers (Ted Pella, Reading, CA, USA), air dried and observed with a high resolution in-lens secondary electron detector through a FESEM Zeiss Merlin (Zeiss, Oberkochen, Germany), operating at 2 kV.
Analysis of cell internalization
HeLa cells were cultured at 37 °C in a humidified atmosphere with 5 % CO2 for 24 h and plated in treated 24 well plates (Nunclon surface, Nunc 150628) with MEM-α medium supplemented with 10 % fetal bovine serum (FBS) and 2 mM Glutamax (Gibco, Rockville, MD, USA). Later, medium was removed and the cells were washed with Dulbecco's phosphate-buffered saline (DPBS). Then, 25 nM of T22-GFP-H6 in 250 µl Optipro supplemented with 2 mM l-glutamine was added. After incubation at 37 °C for 1 h, cells were treated with trypsin in DPBS (1 mg/ml) for 15 min, centrifuged at 300×g for 15 min, and pellets were resuspended in 300 µl DPBS. Samples were analyzed by flow cytometry using a FACSCanto system (Becton–Dickinson, Franklin Lakes, NJ, USA) with a 15 W air-cooled argon-ion laser at 488 nm excitation for GFP.
HeLa cells were plated on a MatTek culture dish (MatTek Corporation, Ashland, MA, USA) at 150,000 cells/plate and incubated at 37 °C for 24 h. After incubation, medium was removed to wash the cells twice with DPBS. Optipro medium supplemented with 2 mM l-glutamine and 25 nM T22-GFP-H6 were added, and cells were incubated at 37 °C for 1 h. To visualize the samples, plasma membranes and the nucleus were labeled using 2.5 mg/ml CellMask Deep Red and 0.2 mg/ml Hoechst 33342 (Molecular Probes, Eugene, OR, USA), respectively. A confocal laser scanning microscope TCS-SP5 (Leica Microsystems, Heidelberg, Germany) was used to analyze the samples, and the images were processed through the Imaris Bitplane software (Bitplane, Zurich, Switzerland).
Determination of in vivo biodistribution
This study was approved by the Institutional Animal Ethics Committee. We implanted CXCR4-overexpressing SP5 human colorectal tumor line to generate subcutaneous colorectal tumors in swiss nude mice (Charles River, France) as previously described [16, 17]. When tumors reached ca. 500 mm3, mice were randomly allocated to Origami B, MC4100, KPM335, BW30270 or buffer-treated groups (N = 3–5/group). The experimental mice received a single intravenous bolus of the corresponding nanoparticle (500 μg in carbonate buffer, pH 7.4), whereas control mice received only buffer. Five hours after the administration, we measured ex vivo the fluorescence emitted by the nanoparticles accumulated in the whole and slice sectioned tumor and normal tissues (kidney, lung, and heart, liver and brain) using IVIS® Spectrum equipment (Perkin Elmer, Waltham, MA, USA). The fluorescence signal was digitalized, and after subtracting the autofluorescence, it was displayed as a pseudocolor overlay and expressed as Radiant efficiency. Data were corrected by the specific fluorescence emitted by the different nanoparticles. Signal difference between groups was determined by applying the non-parametric Mann–Whitney test.
Histological and immunohistochemical evaluation
At necropsy, tumors were fixed with 4 % formaldehyde in PBS for 24 h and embedded in paraffin for histological and immunohistochemical evaluation. 4 µm sections, were processed as previously described [16, 17] and haematoxylin and eosin (H&E) stained for histological analysis, that was performed by two independent observers. We assessed CXCR4 membrane expression and nanoparticle cell internalization in tumor and normal tissues by IHC using primary anti-CXCR4 (1:300; Abcam, Cambridge, UK) or anti-GFP (1:100; Santa Cruz Biotechnology, CA, USA), and secondary HRP conjugated antibody, followed by chromogenic detection. We quantified the percentage of CXCR4-expressing cells in relation to the total cell number and their staining intensity, scoring each sample from 0 to 3 (where 3 is the maximal intensity) and multiplying both parameters to obtain its H-score. Representative pictures were taken using CellэB software (Olympus Soft Imaging v3.3, Tokyo, Japan) at 400×.
Quantitative real-time PCR
Primers used for RT-qPCR
Amplicon length (bp)
Results and discussion
In a previous study , we have demonstrated that mutations in the E. coli genome that affect protein quality control activities (chaperones and proteases) impact on the architecture and function of produced protein nanoparticles . Also, the growth of cultured KPM335 is slightly slower than that of the parental strain BW30270 . Reduced bacterial growth, as promoted, for instance, by minimal defined media or by entering into the stationary phase, results in the modification of intracellular levels of chaperones such as increased levels of ClpB and IbpA and reduced levels of GrpE, HtpG and Hsc . In this context, we wondered if the LPS engineering of KPM335 might have resulted in a physiological, indirect impact on protein processing by a modified cell factory through modification of chaperone balance. Transcriptomic analysis of main folding-assistant genes in KPM335 revealed significant differences regarding the parental BW30270 (Fig. 8b), and in some cases a pattern of expression that might be comparable to that found in proteomics of wild-type bacteria at the end of the exponential growth phase (Fig. 8b). In particular, tig transcript levels are lower being the production of trigger factor associated with ribosomal protein expression and growth rate . On the other hand, some heat shock protein genes are upregulated, in particular those involved in inclusion body disaggregation (clpB and ibpA) [27, 28] indicating enhanced aggregation in the endotoxin-free strain when comparing with the parental background. In this regard, the formation of inclusion bodies by an aggregation-prone protein is slightly favoured in KPM335 when compared to BW30270, as measured by the percentage of insoluble recombinant protein over the total protein yield (86.7 ± 1.2 versus 81.649 ± 1.4 %; data calculated from ). Also, ClpB and IbpA also respond, independently of protein production, to growth rate. They are present in higher amounts in stationary phase at least when cells are growing in complex medium and the growth rate declines gradually . On the other hand, some heat shock protein genes are downregulated (e.g. grpE), that is again in agreement with the endotoxin-free strain growing slower, as there is a slight tendency that these heat shock proteins do not change with growth rate or they are present in lower amounts when the cells grow slower or approach stationary phase .
In summary, the set of seven different mutations in LPS biosynthesis genes of KPM335 promotes a reduction of the cell growth rate that impact, intrinsically, on the expression of several among the main chaperones of the E. coli quality control network. This may have indirect influences on the conformational status of recombinant aggregation-prone protein materials and their building blocks. Although a more complete systems level analysis of KPM335 performance as a cell factory would be desirable, the set of data presented here indicates that LPS engineering secondarily affects the arm of the cells' biosynthetic and maintenance machineries dealing with protein conformation. While this fact does not negatively affect the final quality (tumor-homing) and organization (self-assembling) of complex smart materials such as CXCR4-targeted protein nanoparticles, it indeed influences some of their relevant properties at the macroscopic level.
FR performed most of the experiments regarding protein production and in vitro characterization. MVC performed the in vivo experiments and its linked analysis while AS-C executed the electron microscopy observations, JS-F parts of confocal microscopy and flow cytometry, MP the CD analysis, UU protein purification and UM the quantitative PCR. NF-M and EV designed most of the experimental set-up, UR analysed and discussed the proteomics and transcriptomics data, RM coordinated the in vivo experimental while EG-F and AV conceived the whole study. All authors have participated in the preparation of the manuscript (text and figures), that has been mostly written by AV. All authors read and approved the final manuscript.
Protein production has been partially performed by the ICTS “NANBIOSIS”, more specifically by the Protein Production Platform of CIBER in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN)/IBB, at the UAB (http://www.nanbiosis.es/unit/u1-protein-production-platform-ppp/). We are indebted to MINECO BIO2013-41019-P, AGAUR (2014SGR-132) and CIBER de Bioingeniería, Biomateriales y Nanomedicina (project NANOPROTHER) to AV, Plan Estatal de I+D+I 2013-2016 del Instituto de Salud Carlos III (FEDER co-funding) FIS PI12/00327 to EV, Marató TV3 416/C/2013-2030 to RM for funding our research. We thank the CIBER-BBN Nanotoxicology Unit for fluorescent in vivo follow-up using the IVIS equipment. The authors thank Manuel Hein and Dörte Grella (Research Center Borstel) and also Fran Cortes the Cell Culture and Citometry Units of the Servei de Cultius Cel·lulars, Producció d’Anticossos i Citometria (SCAC), Servei de Proteómica and to the Servei de Microscòpia, for technical assistance, and the Soft Materials Service (ICMAB-CSIC/CIBER-BBN). FR and MP acknowledge financial support from “Francisco Jose de Caldas” Scholarship program of COLCIENCIAS (Colombia) and Universitat Autònoma de Barcelona through pre-doctoral fellowships respectively. UU received a Sara Borrell postdoctoral fellowship from ISCIII. AV received an ICREA ACADEMIA award. Strain KPM335 was kindly provided by Research Corporation Technologies, Tucson, AZ.
UU, EV, MVC, AV, NF-M and RM are co-authors of a patent (WO2012095527) covering the use of T22 as an intracellular targeting agent. No other potential competing interests have been identified.
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