A novel expression system for production of soluble prion proteins in E. coli
© Abskharon et al; licensee BioMed Central Ltd. 2012
Received: 22 July 2011
Accepted: 10 January 2012
Published: 10 January 2012
Expression of eukaryotic proteins in Escherichia coli is challenging, especially when they contain disulfide bonds. Since the discovery of the prion protein (PrP) and its role in transmissible spongiform encephalopathies, the need to obtain large quantities of the recombinant protein for research purposes has been essential. Currently, production of recombinant PrP is achieved by refolding protocols. Here, we show that the co-expression of two different PrP with the human Quiescin Sulfhydryl OXidase (QSOX), a human chaperone with thiol/disulfide oxidase activity, in the cytoplasm of E. coli produces soluble recombinant PrP. The structural integrity of the soluble PrP has been confirmed by nuclear magnetic resonance spectroscopy, demonstrating that properly folded PrP can be easily expressed in bacteria. Furthermore, the soluble recombinant PrP produced with this method can be used for functional and structural studies.
Prion diseases, also referred as transmissible spongiform encephalopathies (TSEs), are a family of rare progressive neurodegenerative disorders that affect both humans and animals . TSEs include, for instance, bovine spongiform encephalopathy (BSE) in cattle, and Creutzfeldt-Jakob disease (CJD) in humans. These disorders are characterized by long incubation periods and characteristic spongiform changes in the brain associated with neuronal loss. The causative agent of TSEs is an infectious protein known as prion (also denoted as PrPSc) . This pathogenic beta-sheet-rich conformer derives from the normal, mostly alpha-helical isoform, cellular prion protein (PrP or PrPC), through a conformational conversion event which leads to aggregates in the brains of affected individuals leading to neurodegeneration .
Since the identification of prions as the causing agent of TSEs, recombinant PrP has been instrumental to study the structural and biophysical aspects of prion amyloidosis .
Due to its easy handling, inexpensive medium and large-scale production, the enteric bacterium Escherichia coli (E. coli) is the organism of choice for the production of numerous recombinant proteins . However, expression of mammalian proteins in E. coli remains difficult and often results in inactive aggregates because the recombinant proteins do not fold properly in this host . For example, recombinant PrP is largely expressed as inclusion bodies . Most refolding protocols require a large amount of reagents and are time consuming. Success is highly dependent on the experimenter's savoir-faire. Attempts to assist proper folding of the PrP in the cytoplasm of E. coli by co-expression with bacterial chaperones failed . As the formation of a disulfide bond is essential for PrP proper folding, expression of full-length PrP in the periplasm of E. coli was also investigated, resulting in soluble PrP which is partially degraded at the unstructured N-terminal end . It has been observed that PrP can interact with several chaperones from the endoplasmic reticulum (ER) , including Pdia3 (also known as ERp57) and Grp58 (ERp60) , suggesting that in physiological condition, PrP requires assistance to fold into the correct conformation. In addition, PrP contains a disulfide bond which is crucial for the proper α-helical fold . Based on these observations, we investigated the use of QSOX as a folding catalyst for PrP in the cytoplasm of E. coli. QSOX is a human chaperone that introduces disulfide bonds in secreted proteins downstream of the ER , and has been shown to be enzymatically active in the bacterial cytoplasm .
In the present study, we describe for the first time the production of soluble PrP of both mouse (MoPrP) and human (HuPrP) using co-expression with QSOX in the E. coli cytoplasm.
Materials and methods
Human PrP (HuPrP): full length (23-231) and truncated (90-231)
Cloning of HuPrP(90-231) into pET-28a (Novagen) was performed as described previously . HuPrP(23-231) was subcloned from the plasmid HuPrP(23-231)/pET-11a  into pET-28a as BamHI-NdeI fragment using the standard molecular biology techniques.
Mouse PrP(MoPrP): full length (23-230) and truncated (89-230)
Cloning of MoPrP(23-230) and MoPrP(89-230) as described previously .
The human QSOX plasmid was a generous gift from Prof. Colin Thorpe .
Small-scale expression trials
Each PrP construct and the QSOX plasmid were co-transformed into E. coli Rossetta (DE3) pLysS and plated on LB-agar supplemented with 100 μg/mL ampicillin and 25 μg/mL kanamycin. A single colony was used to inoculate a 25 mL pre-culture (LB medium supplemented with 100 μg/mL ampicillin and 25 μg/mL kanamycin). The following day, a 25 mL culture was inoculated with 1 mL of the pre-culture. Cells were induced at A600 = 0.7 by adding 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG). After induction culture was incubated overnight (16 h) at 15°C. Harvested cells were resuspended in lysis buffer: 0.1 g of cell paste/mL of 50 mM potassium phosphate, pH 7.5, 300 mM NaCl supplemented with 0.1 mg/mL lysozyme, 0.1 mg/mL ABESF and 1 μg/mL leupeptin. The lysate was sonicated 4 times, each 30 s at 4°C and was subsequently centrifuged 20 min at 18,000 g. Supernatant was collected and pellet was resuspended in initial volume using lysis buffer. Total fraction, supernatant and pellet were analyzed for the presence of soluble PrP by SDS/PAGE and immunoblotting.
Growth curve and quantification of PrP production
For plotting the growth curve of MoPrP(89-230) small-scale cultures (MoPrP/QSOX and MoPrP/no QSOX) were produced as described previously. Samples were collected at 30 minutes time intervals and A600 was measured. Optical density was plotted versus time.
To quantify the amount of PrP produced at the different times during the growth, 1 L culture was induced as described earlier and 40 mL samples were collected at 0, 1, 2, 4, 8 and 16 h after induction. Cells were collected by centrifugation at 15,000 g for 10 min, weighted and resuspended in (0.1 g of cell paste/mL) volume of lysis buffer to normalize the cell content for each time point. For estimating the total prion production 4 μl were mixed with 1 μl SDS loading buffer (5X) and boiled for 5 min. To determine the quantity of soluble expressed PrP, resuspended cells were lysed by sonication and centrifuged at 18,000 g for 20 min. 4 μl of supernatant was mixed with 1 μl SDS loading buffer (5X) and boiled for 5 min. Collected samples were analyzed on SDS-PAGE and by immunoblotting. Intensity of the blot signals was quantified using the software LabImage 1D Gel Analysis (Kapelan GmbH, Germany).
Mouse monoclonal anti-His antibody was purchased from Sigma Aldrich, Belgium. Proteins analyzed on SDS-PAGE were transferred to nitrocellulose membranes (MACHEREY-NAGEL) and bands were visualized by goat anti-mouse IgG, alkaline phosphatase conjugate (Sigma) using NBT/BCIP as substrate (Roche Diagnostics, GmbH, Germany).
Large-scale protein expression and purification
MoPrP(89-230), HuPrP(90-231), MoPrP(23-230) and HuPrP(23-231) were co-expressed with QSOX in E. coli Rossetta (DE3) pLysS. Expression up-scaling was carried out as follows. Pre-cultures (25 mL) were grown overnight at 37°C in LB medium supplemented with 100 μg/mL ampicillin and 25 μg/mL kanamycin. 10 mL of pre-culture were used to inoculate 1 L of LB medium supplemented with ampicillin and kanamycin. Cells were induced at A600 = 0.7 by adding 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG) and temperature was shifted to 15°C for 16 h.
Cells were harvested by centrifugation (15 min at 15,000 g). Cells pellets were re-suspended as 0.1 g of cell paste/mL in 50 mM potassium phosphate, pH 7.5, 300 mM NaCl supplemented with 0.1 mg/mL lysozyme, 0.1 mg/mL AEBSF and 1 μg/mL leupeptin. Cells were disrupted two times with a French press (10,000 psi) and followed by centrifugation at 4°C for 60 min at 40,000 g. The collected supernatant was loaded on a 5 mL Histrap Ni-NTA column (GE-healthcare) previously equilibrated with 50 mM potassium phosphate pH 7.5, 300 mM NaCl, 10 mM imidazole. The column was washed with five column volumes (CV) of washing buffer: 50 mM potassium phosphate pH 7.5, 1 M NaCl, 50 mM imidazole, followed by ten CV of 50 mM potassium phosphate pH 6.0, 1 M NaCl, 50 mM imidazole. The protein was eluted with a gradient of imidazole from 50 mM to 1 M in 50 mM potassium phosphate pH 7.5. The elution peak fractions were loaded on a SDS/PAGE to evaluate purity, then pooled, and concentrated for a second purification step. Size exclusion chromatography was performed in 20 mM Tris-HCl pH 7.5 with 150 mM NaCl on a Superdex75 HR 10/30 (GE Healthcare). The elution peak was again loaded on SDS/PAGE and collected for a dialysis against 10 mM sodium acetate pH 4.6, 1 mM EDTA followed by the final dialysis buffer of 10 mM sodium acetate pH 4.6. Protein aliquots were stored at -80°C until further usage.
Circular dichroism (CD) experiments
CD spectra were recorded at 25°C using a spectropolarimeter (Jasco, model 715, Tokyo, Japan). The purified protein was diluted in water to the final concentration of 0.2 mg/mL. CD spectra were acquired at a scan speed of 50 nm/min, 1 nm bandwidth and a response time of 1 s. A 0.01 cm path length quartz cells was used to record spectra of the different proteins in the far ultraviolet region (190-260 nm), each spectrum was recorded 4 times. The sample and the buffer solutions were purged with dry nitrogen.
Nuclear magnetic resonance (NMR) measurements
Uniformly 15N and both 15N and 13C-labeled MoPrP(89-230) were produced in M9 minimal medium supplemented with 15NH4Cl and [13C] glucose as described by Marley et al. . The labelled proteins were purified as described above for unlabelled proteins.
The NMR experiments were recorded on 15N, 13C-labeled MoPrP(89-230) concentrated to approximately 0.5 mM into 10 mM sodium acetate buffer pH 4.6. Two dimensional NMR 15N and 13C heteronuclear single quantum correlation (HSQC) spectra and three dimensional NMR experiments CBCA(CO)NH, HNCACB, HNCO, HBHA(CO)NH were performed at 293 K on MoPrP(89-230) on a Varian NMR Direct-Drive Systems 800 MHz spectrometer equipped with a salt tolerance triple-resonance PFG-Z cold probe. Sequence specific backbone 1HN, 15N, 13C', 13Ca, 13Cb, Ha and Hb chemical shifts were determined using standard triple-resonance assignment methodology . Identical experiments were performed for MoPrP(89-230) at pH 7.0. All the NMR data were processed with NMRPipe 2.1 software  and analysed using CCPNmr Analysis 2.0 . CS23D2.0  was used to generate a 3D model of MoPrP(89-230) at pH 4.6 using only the backbone chemical shifts.
The thiostar assay determines accurately the amount of free thiol content in samples . To perform this assay, we used the kit Detect X™, Luminos (Arbor assays, USA). A standard curve was plotted using reduced L-glutathion (Sigma, Belgium) at different concentrations (0 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM and 60 μM). Two protein samples were tested: soluble PrP and refolded, both samples were tested at a fixed concentration of 5 μM. All standards and samples were diluted 10 times in a thiol-free buffer to a final volume of 100 μL in a 96-well plate. Then we added 15 μL of Thiostar reagent to each well. After mixing, we incubated the plate at room temperature for 30 min in the dark. The fluorescent product was measured at 510 nm in a fluorescent plate-reader (Infinite M200, TECAN) with excitation at 390 nm.
Amyloid seeding assay (ASA) protocol
MoPrP(89-230) has been diluted to 0.1 mg/mL in phosphate-buffered saline (PBS) solution containing 0.4 M Guanidine hydrochloride (GuHCl), 10 mM Thioflavin T (ThT). The fibrilization reaction was performed in a final volume of 200 μL in 96-well plate (BD Falcon, BD Bioscience). For seeding experiment we performed the amyloid seeding assay (ASA) according to Colby et al., 2007  with some minor modifications. Briefly, 1 mg of ScGT1 cell lysate were used for PTA precipitation by adding 500 μL of PBS containing 4% sarkosyl, protease inhibitor (Complete, Roche) and 0.5% PTA, with continuous shaking at 37°C, 350 rpm for 1 h and centrifuged 14,000 g for 30 min. The pellet was washed and resuspended in the previous buffer, then centrifuged again and re-suspended in 150 μL of sterile double distilled H2O. In ASA, 4 μL of re-suspended PTA pellet were diluted in 400 μL of water and 20 μL of diluted sample were added to each well. The plate was incubated at 37°C with continuous shaking on a plate reader (Spectramax M5, Molecular Device). The kinetics of fibril formation was monitored by top reading of fluorescence intensity every 5 min at 444 nm excitation and 485 nm emission.
Conversion of the monomeric MoPrP into amyloid fibrils
MoPrP(89-230) was diluted into 50 mM phosphate buffer, 2 M GuHCl, pH 6.5 to a final concentration of 0.4 mg/ml and incubated at 37°C with constant rotation (8 rpm).
Atomic Force Microscopy (AFM)
The converted PrP samples were incubated on a freshly cleaved mica surface for approximately 30 seconds and subsequently rinsed with water to remove salts and unbound protein. After drying the samples were imaged using tapping mode on a Digital Instruments Multimode atomic force microscope equipped with Nanoscope IV controller and a type E scanner. All images were acquired using single-beam silicon probes with a nominal spring constant of 40 N/m and nominal tip radius of 10 nm.
X-ray fiber diffraction
Converted PrP was pelleted by centrifugation at 16,000 g, and pellet was subsequently washed twice with water to remove salts. The wet pelleted fibrils were pipetted in a 2 mm space between two fire-polished glass rods and allowed to dry. Diffraction patterns were collected using a Rigaku MicroMax-007HF copper anode X-ray source and a Rigaku Saturn 944+ CCD detector.
Results and discussion
The formation of the disulfide bond between Cys179 and Cys214 is essential to obtain a correctly folded PrP [12, 32]. The human Quiescin Sulfhydryl OXidase (QSOX) allows the formation of native disulfide bonds in eukaryotic proteins expressed in the cytoplasm of E. coli [14, 33]. We showed that co-expressing PrP (full-length or truncate MoPrP and HuPrP) with QSOX produces a significant amount of correctly folded PrP in the cytoplasm and that a disulfide bond is present in the purified protein. Previous studies showed that co-expression with several chaperones did not succeed in producing reasonable quantities of soluble PrP . Furthermore, soluble but partially degraded PrP could only be produced in the periplasm . In E. coli, disulfide bond formation occurs in the periplasm . Co-expression with QSOX is a good alternative to produce mammalian proteins containing disulfide bonds in the E. coli.
To the best of our knowledge, there is no evidence for direct interaction in vivo of QSOX with the PrP; therefore we believe that QSOX could be used to express different mammalian PrP in E. coli. The described method is a simple and effective method for producing large quantities of soluble PrP in E. coli, which can subsequently be used for functional and structural studies.
The plasmid containing the human QSOX gene was kindly provided by Prof. Colin Thorpe (University of Delaware). JM is a group leader of the VIB. We would like to thank Prof. Nico van Nuland for NMR assistance.
RNNA is supported by the European student exchange program "Erasmus Mundus ECW II" reference number (132878-EM-1-2007-BE-ERA Mundus-ECW). WG is supported by the European student exchange program "Erasmus Mundus ECW II" reference (141085-EM-1-2008-BE-ERA Mundus-ECW).
- Colby DW, Prusiner SB: Prions. Cold Spring Harb Perspect Biol 2011,3(1):a006833. 10.1101/cshperspect.a006833View ArticleGoogle Scholar
- Prusiner SB: Novel proteinaceous infectious particles cause scrapie. Science 1982,216(4542):136-144. 10.1126/science.6801762View ArticleGoogle Scholar
- Watts JC, Balachandran A, Westaway D: The expanding universe of prion diseases. PLoS Pathog 2006,2(3):e26. 10.1371/journal.ppat.0020026View ArticleGoogle Scholar
- Soto C: Prion hypothesis: the end of the controversy? Trends Biochem Sci 2011,36(3):151-158. 10.1016/j.tibs.2010.11.001View ArticleGoogle Scholar
- Makrides SC: Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Rev 1996,60(3):512-538.Google Scholar
- Baneyx F, Mujacic M: Recombinant protein folding and misfolding in Escherichia coli. Nat Biotechnol 2004,22(11):1399-1408. 10.1038/nbt1029View ArticleGoogle Scholar
- Mehlhorn I, Groth D, Stockel J, Moffat B, Reilly D, Yansura D, Willett WS, Baldwin M, Fletterick R, Cohen FE, et al.: High-level expression and characterization of a purified 142-residue polypeptide of the prion protein. Biochemistry 1996,35(17):5528-5537. 10.1021/bi952965eView ArticleGoogle Scholar
- Kyratsous CA, Silverstein SJ, DeLong CR, Panagiotidis CA: Chaperone-fusion expression plasmid vectors for improved solubility of recombinant proteins in Escherichia coli. Gene 2009, 440:(1-2):9-15. 10.1016/j.gene.2009.04.002View ArticleGoogle Scholar
- Hornemann S, Glockshuber R: Autonomous and reversible folding of a soluble amino-terminally truncated segment of the mouse prion protein. Journal of molecular biology 1996,261(5):614-619. 10.1006/jmbi.1996.0487View ArticleGoogle Scholar
- Watts JC, Huo H, Bai Y, Ehsani S, Jeon AH, Shi T, Daude N, Lau A, Young R, Xu L, et al.: Interactome analyses identify ties of PrP and its mammalian paralogs to oligomannosidic N-glycans and endoplasmic reticulum-derived chaperones. PLoS Pathog 2009,5(10):e1000608. 10.1371/journal.ppat.1000608View ArticleGoogle Scholar
- Hetz C, Russelakis-Carneiro M, Walchli S, Carboni S, Vial-Knecht E, Maundrell K, Castilla J, Soto C: The disulfide isomerase Grp58 is a protective factor against prion neurotoxicity. J Neurosci 2005,25(11):2793-2802. 10.1523/JNEUROSCI.4090-04.2005View ArticleGoogle Scholar
- Maiti NR, Surewicz WK: The role of disulfide bridge in the folding and stability of the recombinant human prion protein. J Biol Chem 2001,276(4):2427-2431. 10.1074/jbc.M007862200View ArticleGoogle Scholar
- Heckler EJ, Alon A, Fass D, Thorpe C: Human quiescin-sulfhydryl oxidase, QSOX1: probing internal redox steps by mutagenesis. Biochemistry 2008,47(17):4955-4963. 10.1021/bi702522qView ArticleGoogle Scholar
- Nguyen VD, Hatahet F, Salo KE, Enlund E, Zhang C, Ruddock LW: Pre-expression of a sulfhydryl oxidase significantly increases the yields of eukaryotic disulfide bond containing proteins expressed in the cytoplasm of E.coli. Microbial cell factories 2011, 10: 1. 10.1186/1475-2859-10-1View ArticleGoogle Scholar
- Abskharon RN, Soror SH, Pardon E, El Hassan H, Legname G, Steyaert J, Wohlkonig A: Combining in-situ proteolysis and microseed matrix screening to promote crystallization of PrPc-nanobody complexes. Protein Eng Des Sel 2011,24(9):737-741. 10.1093/protein/gzr017View ArticleGoogle Scholar
- Kosmac M, Koren S, Giachin G, Stoilova T, Gennaro R, Legname G, Serbec VC: Epitope mapping of a PrP(Sc)-specific monoclonal antibody: identification of a novel C-terminally truncated prion fragment. Mol Immunol 2011,48(5):746-750. 10.1016/j.molimm.2010.11.012View ArticleGoogle Scholar
- Marley J, Lu M, Bracken C: A method for efficient isotopic labeling of recombinant proteins. J Biomol NMR 2001,20(1):71-75. 10.1023/A:1011254402785View ArticleGoogle Scholar
- Bottomley MJ, Macias MJ, Liu Z, Sattler M: A novel NMR experiment for the sequential assignment of proline residues and proline stretches in 13C/15N-labeled proteins. J Biomol NMR 1999,13(4):381-385. 10.1023/A:1008393903034View ArticleGoogle Scholar
- Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A: NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 1995,6(3):277-293.View ArticleGoogle Scholar
- Vranken WF, Boucher W, Stevens TJ, Fogh RH, Pajon A, Llinas M, Ulrich EL, Markley JL, Ionides J, Laue ED: The CCPN data model for NMR spectroscopy: development of a software pipeline. Proteins 2005,59(4):687-696. 10.1002/prot.20449View ArticleGoogle Scholar
- Wishart DS, Arndt D, Berjanskii M, Tang P, Zhou J, Lin G: CS23D: a web server for rapid protein structure generation using NMR chemical shifts and sequence data. Nucleic Acids Res 2008, (36 Web Server):W496-502.Google Scholar
- Park S, Lippard SJ: Redox state-dependent interaction of HMGB1 and cisplatin-modified DNA. Biochemistry 2011,50(13):2567-2574. 10.1021/bi2000214View ArticleGoogle Scholar
- Colby DW, Zhang Q, Wang S, Groth D, Legname G, Riesner D, Prusiner SB: Prion detection by an amyloid seeding assay. Proc Natl Acad Sci USA 2007,104(52):20914-20919. 10.1073/pnas.0710152105View ArticleGoogle Scholar
- Flores S, de Anda-Herrera R, Gosset G, Bolivar FG: Growth-rate recovery of Escherichia coli cultures carrying a multicopy plasmid, by engineering of the pentose-phosphate pathway. Biotechnol Bioeng 2004,87(4):485-494. 10.1002/bit.20137View ArticleGoogle Scholar
- Gatti-Lafranconi P, Natalello A, Ami D, Doglia SM, Lotti M: Concepts and tools to exploit the potential of bacterial inclusion bodies in protein science and biotechnology. FEBS J 2011,278(14):2408-2418. 10.1111/j.1742-4658.2011.08163.xView ArticleGoogle Scholar
- Wishart DS, Sykes BD: The 13C chemical-shift index: a simple method for the identification of protein secondary structure using 13C chemical-shift data. J Biomol NMR 1994,4(2):171-180.View ArticleGoogle Scholar
- Riek R, Wider G, Billeter M, Hornemann S, Glockshuber R, Wuthrich K: Prion protein NMR structure and familial human spongiform encephalopathies. Proc Natl Acad Sci USA 1998,95(20):11667-11672. 10.1073/pnas.95.20.11667View ArticleGoogle Scholar
- Polano M, Bek A, Benetti F, Lazzarino M, Legname G: Structural insights into alternate aggregated prion protein forms. Journal of molecular biology 2009,393(5):1033-1042. 10.1016/j.jmb.2009.08.056View ArticleGoogle Scholar
- Ai Tran HN, Sousa F, Moda F, Mandal S, Chanana M, Vimercati C, Morbin M, Krol S, Tagliavini F, Legname G: A novel class of potential prion drugs: preliminary in vitro and in vivo data for multilayer coated gold nanoparticles. Nanoscale 2010,2(12):2724-2732. 10.1039/c0nr00551gView ArticleGoogle Scholar
- Wille H, Bian W, McDonald M, Kendall A, Colby DW, Bloch L, Ollesch J, Borovinskiy AL, Cohen FE, Prusiner SB, et al.: Natural and synthetic prion structure from X-ray fiber diffraction. Proc Natl Acad Sci USA 2009,106(40):16990-16995. 10.1073/pnas.0909006106View ArticleGoogle Scholar
- Sunde M, Serpell LC, Bartlam M, Fraser PE, Pepys MB, Blake CC: Common core structure of amyloid fibrils by synchrotron X-ray diffraction. Journal of molecular biology 1997,273(3):729-739. 10.1006/jmbi.1997.1348View ArticleGoogle Scholar
- Welker E, Wedemeyer WJ, Narayan M, Scheraga HA: Coupling of conformational folding and disulfide-bond reactions in oxidative folding of proteins. Biochemistry 2001,40(31):9059-9064. 10.1021/bi010409gView ArticleGoogle Scholar
- Thorpe C, Coppock DL: Generating disulfides in multicellular organisms: emerging roles for a new flavoprotein family. J Biol Chem 2007,282(19):13929-13933. 10.1074/jbc.R600037200View ArticleGoogle Scholar
- de Marco A: Strategies for successful recombinant expression of disulfide bond-dependent proteins in Escherichia coli. Microbial cell factories 2009, 8: 26. 10.1186/1475-2859-8-26View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.