Nova Biotechnol Chim (2017) 16(1): 1-11 DOI: 10.1515/nbec-2017-0001  Corresponding author: jamrichova.daniela@gmail.com, lenka.tisakova@ucm.sk Nova Biotechnologica et Chimica How to approach heterogeneous protein expression for biotechnological use: An overview Daniela Jamrichová, Lenka Tišáková, Veronika Jarábková and Andrej Godány Department of Biology, Faculty of Natural Sciences, University of SS. Cyril and Methodius in Trnava, Nám. J. Herdu 2, Trnava, SK-917 01, Slovak Republic Article info Article history: Received: 8th June 2017 Accepted: 26th June 2017 Keywords: Escherichia coli Gene expression Protein purification Heterologous Abstract Production of recombinant proteins in Escherichia coli expression systems has shown many advantages, as well as disadvantages, especially for biotechnological and other down-stream applications. The choice of an appropriate vector depends on the gene, to be cloned for purification procedures and other analyses. Selection of a suitable production strain plays an important role in the preparation of recombinant proteins. The main criteria for the selection of the host organism are the properties of the recombinant produced protein, its subsequent use and the total amount desired. The most common problems in eukaryotic gene expression and recombinant proteins purification are, for instance, post-translational modifications, formation of disulphide bonds, or inclusion bodies. Obtaining a purified protein is a key step enabling further characterization of its role in the biological system. Moreover, methods of protein purification have been developed in parallel with the discovery of proteins and the need for their studies and applications. After protein purification, and also between the individual purification steps, it is necessary to test protein stability under different conditions over time. Shortly, all the essential points have been briefly discussed, which could be encountered during production and purification of a recombinant protein of interest, especially from eukaryotic source and expressed heterogeneously in prokaryotic production system.  University of SS. Cyril and Methodius in Trnava Introduction Over years, the production of recombinant proteins in the Escherichia coli expression system has shown many advantages, as well as disadvantages. There is the evidence of such recombinant protein evolution reflecting in a wide range of commercially available production Escherichia coli strains with modified properties for the heterogeneous production of eukaryotic proteins under various conditions. The major advantage of an expression system with E. coli as a host organism is time, technical and financial effectivity. E. coli cells can be propagated under common laboratory conditions in a simple culture medium with selected ions, salts and carbon sources. In the exponential phase, its generation time is 20 min, in a complex culture medium containing vitamins, salts, microelements, amino acids and glucose, at an intensive passage at 37 °C (Sezonov et al. 2007). It is doubtless that the production of recombinant proteins in various microbial systems (especially those of eukaryotic origin), has revolutionized the whole biochemical and biotechnological overview of the basic approach how to obtain high yield of protein Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 27.02.20 14:22 UTC mailto:jamrichova.daniela@gmail.com Nova Biotechnol Chim (2017) 16(1): 1-11 2 in a decent purity. Many scientific studies focused on application output of proteins have encountered obstacles with heterogeneous expression of eukaryotic genes in prokaryotic systems, because of miscellaneous reasons. This paper could serve as brief overview that pays attention to crucial steps and potential hinders in production of eukaryotic recombinant proteins in heterogeneous E. coli, methods of isolation, purification and detection of the (overexpressed) recombinant proteins, especially (Fig. 1). Gene design When planning production of recombinant protein, it is inevitable to take into consideration several steps in preparation of the gene cloned. Principally, before starting the experimental part, it is important to apply bioinformatics approaches to evaluate the usability/suitability of the gene of interest for the planned downstream procedures. Various in silico analyses could be conducted by online programs and tools, such as restriction digestion analysis of the gene, primer design for amplification of the gene, primer melting temperature calculation, verification the open reading frames (ORFs). It should be emphasized that E. coli codon usage evaluation is one of the key factors for heterogeneous expression, so it must be performed prior to gene design. Nowadays, several commercial companies provide codon usage optimization as one of their bonus services along with gene synthesis in a selected type of vector. In the case of using a synthetic gene for recombinant protein production, much time could be saved in cloning procedures and the real starting point in the laboratory is the expression part. Last but not least, when thinking about fusion protein preparation, it is good to evaluate, whether to design a (synthetic) gene with or without a stop codon. Finally, hasty decision while designing a gene, cloning the gene or directly ordering a synthetic one, could rapidly decrease the rate of getting relevant results and increase the sources needed to reassess or repeat the procedures. Fig. 1. Overview of the steps (in blocks) for heterogeneous protein production with all the essential steps involved – obstacles during protein production (bars above blocks) and their consequences (bars below the corresponding blocks). Vector selection The choice of an appropriate vector for successful down-stream procedures depends on the gene to be cloned. All expression vectors must contain three important signal sequences including promoter, terminator and ribosome binding site (RBS). Currently, the most common expression vectors are designed as a combination of replicon, promoter, RBS, selection marker, multicloning site (MCS), affinity tag to facilitate purification, and sometimes a fusion protein to increase protein solubility. There is a wide range of commercially available expression vectors, so the selection of a suitable Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 27.02.20 14:22 UTC Nova Biotechnol Chim (2017) 16(1): 1-11 3 vector for a specific gene of interest to be cloned must be not only complex, but also substantial. Therefore, attention should be paid to the needs of the (individual) gene to be cloned and expressed, when searching for the most suitable expression vector. The promoter is a cardinal part of the expression vector, as a promotor sequence regulates the initial stage of gene expression, RNA polymerase attachment, and controls the rate of mRNA synthesis. There are a number of vectors on the market with different types of promoters, while the most commonly used promoter types are listed in Table 1. Escherichia coli expression systems For cloning under laboratory conditions and in industrial scale-up production technologies, non-pathogenic E. coli strains have become the starting point for the developing new strains with features optimized for gene cloning or recombinant protein expression and production. The fundamental task in the heterologous protein production in bacterial expression systems is the correct folding of the produced protein. Since the E. coli production strains lack the systems necessary to handle more complex eukaryotic proteins or to further modify them (e.g. glycosylation), special strains have been developed, e.g. Origami that supports the formation of disulphide bridges in the cytoplasm (Derman et al. 1993) or Rosetta (Novagen, USA) with modified tRNA codes providing easier expression of eukaryotic genes in the prokaryotic systems (Guillaume et al. 2006; Nakamura et al. 2008). Selection of a suitable production strain plays an important role even for following downstream procedures. The main criteria for the selection of the host organism are the properties of the recombinant protein produced, its subsequent use and the total amount. Inappropriate choice of the production strain may cause the recombinant protein to be inactive, insoluble, degraded, or can only be produced in limited amounts. Therefore, E. coli strains have been developed that lack nucleases and proteases, to prevent degradation of the recombinant DNA and the produced protein. A variety of E. coli strains with various desired properties have been developed by mutations of specific genes (Baneyx 1999; Yokoyama 2003; Klock et al. 2005; Terpe 2006). The most common E. coli strains include BL21 (derived from the B strain) and derivatives thereof. BL21 strains are non-pathogenic, capable of growth in simple culture media, allowing higher gene expression under the T7 bacteriophage promoter integrated into the bacterial chromosome of the bacterium (Studier and Moffatt 1986). Nowadays, there are many biotechnologically and therapeutically used proteins produced in E. coli cells (Chrastilová et al. 2007). Selection of a host strain When using E. coli as a production organism for expression of recombinant proteins, some drawbacks could be encountered, directly derived from its inherent properties, especially due to its prokaryotic nature. There are some typical and commonly occurring problems in expressing recombinant genes and producing recombinant proteins. Post-translational modifications Protein post-translational modifications (phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation and proteolysis) increase the functional diversity of the proteome by the covalent addition of functional groups or proteins, proteolytic cleavage of regulatory subunits or degradation of entire proteins. These modifications generally influence almost all aspects of normal cell biology and pathogenesis, therefore, identifying and understanding PTMs it is critical to identify and understand them, when focusing on gene expression and protein production. There are no posttranslational modifications occurring during the protein production in E. coli, such as N-glycosylation, C-glycosylation, acetylation, phosphorylation, which might be indispensable for proper functions of eukaryotic proteins (Stevens 2000; Yin et al. 2007). This problem can be addressed by using evolutionary higher systems, for example, yeasts, or mammalian cells. From another point of view, lack of post-translational modifications can also be Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 27.02.20 14:22 UTC Nova Biotechnol Chim (2017) 16(1): 1-11 4 beneficial, when protein production in non- glycosylated form is more suitable for subsequent applications, for instance, protein crystallisation. Formation of disulphide bonds To ensure catalysis of protein disulphide bonds (Dsb) formation, there have been evolved elaborate Dsb systems in Escherichia coli (Inaba 2009). Disulphide bonds are important for protein structure and functionality but are only formed in the periplasm under catalysis of the Dsb system (Andersen et al. 1997; Bardwell 1994). Therefore, if disulphide bond formation is required, the recombinant protein should be directed to the periplasmic space by means of a cleavable Table 1. Digest of the most commonly used promoters in E. coli expression vectors. signal peptide (e.g. pelB) (Chung et al. 1998; Le-Calvez et al. 1996). Such a signal is used by W3110 production strains as well as by some vectors, for example the pET26b (Makrides 1996; Matthey et al. 1999). The lower abundance of proteins in the periplasm facilitates the recombinant protein purification (Choi and Lee 2004). However, only small yields of the final proteins are usually obtained in the periplasm, which is marked as the major drawback of this type of production. A more convenient approach is the use of specific, commercially available strains, for example Origami strain (Novagen). These strains involve mutated genes encoding for thioredoxine reductase (trxB) and glutathione reductase (Gor) in the Dsb system, allowing cytoplasmic cysteine reduction by thioredoxin and glutathione redoxine (Bessette et al. 1999). If further formation of disulphide bonds is needed, the gene for thioredoxin can by fused with the gene of interest and subsequently expressed in the trxB / gor E. coli strain (Lavallie et al. 1993). If the protein does not get the correct tertiary structure (folding), it is usually insoluble and creates a so-called inclusion bodies within the bacterial host (Chatterjee and Esposito 2006). Formation of inclusion bodies Proteins in inclusion bodies are inactive, aggregated and insoluble. They usually require denaturation and subsequent renaturation, Promotor Type Features References Lac Inducer: IPTG. Lac promoters and their lacUV5 derivatives are weak promoters therefore not often used for recombinant proteins production. Müller-Hill et al. (1968), Calos (1978), Deuschle et al. (1986), Makoff and Oxer (1991) Trp Inducer: indolyl-3-acrylic acid. It is used to produce majority of protein. Hallewell and Emtage (1980), Tacon et al. (1983), Yansura (1990) Tac Inducer: IPTG. A hybrid type of promoter prepared by joining (-35) trp and (-10) lac promoter regions. Tac is 10 fold stronger than lacUV5 promoter. De Boer et al. (1983) λPL Inducer: heat shock. A strong type of promotor with wide use in industrial biotechnology at higher fermentation temperatures. Menart et al. (2003), Dodd et al. (2005), Valdez- Cruz et al. (2010) T7 Inducer: lactose or IPTG. Basal expression is controlled by lacIQ as well as co- expression of T7 lysozyme. Moffatt and Studier (1987), Baneyx (1999), Graumann and Permstaller (2006), Stano and Patel (2004) Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 27.02.20 14:22 UTC Nova Biotechnol Chim (2017) 16(1): 1-11 5 re-folding, which complicates protein production and reduces yields (Graslund et al. 2008). On the other hand, if there is no problem with protein renaturation, the inclusion bodies can be easily separated by centrifugation since they contain only small amounts of contaminants, and ultimately, the higher purity of the recombinant protein (Baneyx and Mujacic 2004; Cabrita and Bottomley 2004). Formation of inclusion bodies can be suppressed by slowing expression of the (trans)gene. This can be achieved in different ways e.g. by reducing the cultivation temperature, using a weaker promoter, application of lower concentrations of inducer. Slowing down the formation of the protein increases the chance for its proper folding. Presence of lipopolysaccharides Another obstacle in the production of proteins in E. coli may be the presence of lipopolysaccharides which constitute a part of the cell wall of gram-negative bacteria. These lipopolysaccharides have pyrogenic effects and cause complications in the purification of end products. Therefore, de-pyrogenation is needed (Terpe 2006; Yin et al. 2007), despite the reduced final yield of the pure protein and possible adverse effect on protein stability and native conformation (Petsch and Anspach 2000). Toxicity of the recombinant proteins Toxic (recombinant) proteins cause death of the host cells or complication during their cultivation. Since the toxicity for the given expression strain can be specific, the strain can be replaced by a more compatible one for protein production. Another option is the use of a highly regulated expression system, where the expression of the gene of interest is regulated by an inducible promoter and transcription terminator. Controlled protein production can result from keeping the plasmid copy number low, while protein toxicity can by modified by engineering the recombinant protein sequence (Saida 2007). Nucleotide sequence of the gene of a foreign origin The eukaryotic gene cloned may contain introns. Since E. coli lacks the necessary splicing mechanism for eukaryotic mRNA, production of active protein poses a serious problem in E. coli. This can be overcome by using complementary DNA (cDNA) prepared from mature mRNA or using a synthetic gene (Le Hir et al. 2003). Purification of recombinant proteins Obtaining a purified protein from expression systems is often a key step enabling its characterization and clarification of its role in biological systems. Purification of recombinant proteins represents a series of procedures that lead to the obtaining of a single protein species from complex bacterial lysates. Methods of protein purification have been developed in parallel with the discovery of proteins and the need for their further study. Until the beginning of the 20th century, filtration, precipitation and crystallization were mainly used to isolate proteins. The breakthrough occurred in 1903 when Cvet separated plant pigments in a calcium carbonate column and subsequently, in 1906, introduced the concept of chromatography for this method. Nearly two decades later, in 1924, Svedberg proved that proteins can be separated by analytical ultracentrifugation (Philo 1997). In the course of several years, other methods of protein separation were discovered, namely electrophoresis, affinity chromatography (AC) and ion exchange chromatography. Since the mid- 20th century, there has been an intensive development of various types of chromatographic matrices. Initially, starch or modified dextran (Sephadex) were only used to allow the separation of proteins by size (Porath and Flodin 1959). To obtain a protein of even a higher purity, other matrices such as polyacrylamide (Raymond and Weintraub 1959), methyl acrylate (Curtain 1963) or agarose (Hjrtén 1964) have been developed that have several advantages over the starch gels like higher stability or easier handling. On the other hand, some disadvantages could emerge, e.g. toxicity of the acrylamide, which comes from a nature of this substance. Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 27.02.20 14:22 UTC Nova Biotechnol Chim (2017) 16(1): 1-11 6 Protein and peptide tags To facilitate detection and purification of the recombinant protein, protein or peptide tags (labels) have been implemented. These tags represent exogenous sequences with high affinities to specific biological or chemical ligands (Bornhorst and Falke 2000; Arnau 2006a). Affinity labelling allows for simple detection of proteins with low immunogenicity or no antibodies available (Brizzard 2008). Protein tags have been proven very suitable in protein purification where the labelled protein can be purified directly from the cell lysate or supernatant (Lichty 2005). Affinity tags may be located at the N- or C-terminus of the recombinant protein (Justesen et al. 2009), for some specific applications it is possible to place the tag even on both ends. The use of tags can improve the biochemical properties and increase the yield of the produced protein, simplify renaturation conditions, increase the solubility of the produced protein, or prevent its proteolysis. However, the use of affinity tags may also have adverse impacts on labelled proteins since, for example, can alter the conformation of the labelled protein, inhibit enzyme activity, Table 2. The most commonly used stabilizing agents for protein storage. Class Examples Value / Usage Buffers Phosphate buffer, Tris-HCl, HEPES, HEPES – NaOH, etc. Stability Salts KCl, NaCl, (NH4)2SO4 Stability Ionic detergents Sodium deoxycholate, Sodium cholate, CTAB, SDS Stability and increase protein solubility Non-ionic detergents Triton X-100, Nonidet P-40, Tween 20, Brij 35, etc. Cryoprotectants Glycerol, Saccharose Storage and stability Chelating agents Citric acid, salicylic acid, EDTA, EGTA Stability Reducing agents β-Mercaptoethanol (BME), dithiotreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP) Stability, prevents formation of disulphide bonds Ligands, cofactors Mg2+, ATP phosphate Stability Protease inhibitors PMSF (phenylmethylsulfonyl fluoride), TPCK (tosyl phenylalanyl chloromethyl ketone), TLCK (tosyl-L-lysyl-chloromethane hydrochloride) Prevents protein degradation cause toxicity, alter biological function and reduce the protein yield (Arnau 2006b). Therefore, when choosing an affinity tag, several inevitable points have to be considered to achieve the desired biochemical features of the protein of interest. These include the bacterial production strain used, the conditions of purification, the desired purity and the final amount of protein. Chromatographic methods for protein purification Presently, various types of liquid chromatography are used to purify recombinant proteins. These differ in the principle of separation of the purified protein and include affinity-, ion exchange-, gel permeation-, or hydrophobic chromatography. Due to the large number of available methods Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 27.02.20 14:22 UTC Nova Biotechnol Chim (2017) 16(1): 1-11 7 and various chromatographic matrices it is necessary to select the purification method prior to the construction of the recombinant molecule. Purification methods based on protein-peptide fusion or on a fusion protein with high affinity for a particular ligand enable using a simple affinity chromatography. Other protein purification methods involve various interactions, such as antibody–antigen, enzyme–substrate, or protein– metal ion. To facilitate purification, modern expression vectors contain specific sequences enabling better detection and/or subsequent purification of expressed proteins, or polypeptide sequences that affect protein solubility. The FLAG was the first tag and represents a hydrophilic sequence of eight amino acids (DYKDDDDK) recognized by a monoclonal antibody. This antibody allows detection of the fusion protein and its purification by immobilization on a suitable chromatographic matrix (Hopp et al. 1988). Currently a histidine tag (His-tag, His6-tag) consisting of 6-12 residues is routinely used for protein purification (Janknecht et al. 1991; Lichty 2005). The high-affinity binding of the imidazole group of histidine to a transition metal like Ni2+, Co2+, Cu2+, Fe2+ or Zn2+is the basis of a method known as Immobilized Metal Affinity Chromatography (IMAC) that has become one of the basic purification procedures for the isolation of recombinant proteins. The metallic ions are immobilized on an agarose or Sepharose matrix by chelation with imidodiacetic acid (IDA), nitrilotriacetic acid (NTA) or tris (carboxymethyl) ethylenediamine (TED) (Porath et al. 1975; Hochuli et al. 1987). Such a matrix is used for purification of His-tagged proteins under both native and denaturation conditions to yield up to ten mg of protein per 1 mL of a matrix. The protein can be eluted from the matrix with buffer containing imidazole (usually 150– 300 mM), EDTA (eluting with metal ions) or by lowering the buffer pH to values in the range 2.5–7.5. The procedure itself and selection of the most suitable metal ion should be optimized separately for each individual protein based on its physicochemical properties (Bolanos-Garcia and Davies 2006). Noteworthy, washing out the metal from the matrix can cause insufficient purity of the protein. This can be avoided using a so called pentadentate (TED) matrix, which binds the metal ions very tightly and thus prevents their leaching, leaving only one free spot for the protein to bind. If affinity chromatography fails to provide the product of the desired purity, other chromatographic methods should be used in subsequent steps, e.g. ion exchange chromatography that separates the molecules by their charge. The stationary phase consists of matrices from dextran derivatives (Sephadex) or agarose (Sepharose) with alkaline functional groups – anionic exchangers, or with acidic functional groups – cationic exchangers. Ion exchange chromatography has been frequently used not only for protein purification but also for its concentrating. Another option for protein purification is a gel permeation chromatography method, also called a molecular sieve, which separates proteins based on their molecular size and also could be used for desalination of protein samples. Monitoring the concentration and purity of proteins Determination of the purity of a recombinant protein is of key importance for its subsequent use. However, a direct method for quantification of the purity of the protein sample is missing. The available procedures always start assessing the amount of individual types of impurities such as of nucleic acids, carbohydrates, lipids, isoenzymes, inactive enzymes. For this operation, the proper type of chemical/physical method should be chosen. The protein purity value is dependent on the type of test method, as well as its sensitivity (detection limit). Therefore, several factors are taken into account when deciding for a particular approach, in particular, the amount of protein available (yield and concentration), the nature of the impurities, and the type of solution, in which the protein is re-suspended. The number of analytical methods for monitoring the purity of proteins include colorimetry, electrophoresis, mass spectrometry, dynamic light scattering and enzyme analysis (Rhodes et al. 2009). Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 27.02.20 14:22 UTC Nova Biotechnol Chim (2017) 16(1): 1-11 8 Storage of purified proteins In terms of maintaining the protein quality required for further procedures, storage of purified proteins is crucial. Storage conditions depend on the type of protein, therefore there is no general rule and procedure available. Importantly, protein samples should not be exposed to extreme changes in pH or temperature, and other factors that could potentially lead to protein denaturation. Long-term storage of the protein solution at room temperature also requires the use of antibacterial and antimycotic agents to avoid biological contamination and destruction of the protein sample. During and after protein purification it is necessary to test the stability of the protein under different conditions, simplest on small aliquots of samples, e.g. at room temperature, at 4–6 °C, at lower temperatures –20 °C, and for its enzymatic activity and concentration over time. Several storage conditions may have an adverse effect on protein stability and activity. An illustrative example is storage at –20°C in 50 % glycerol, which ensures protein stability, however, when followed by purification the glycerol has to be removed from the sample (e.g. by dialysis) to avoid dropping the protein amount and activity. Protein storage is enhanced by rapid freezing without the use of a cryoprotectants (such as glycerol, ethylene glycol, sucrose), however in this case protein freezing and defrosting can cause protein denaturation, aggregation and precipitation (Cao et al. 2003). Proteins can be unstable and lose their biological activity as a result of various physical and chemical procedures, too, even at low temperatures (Carpenter et al. 2002; Simpson 2010). Therefore, when choosing the proper protein storage conditions, it is important to consider what the purified protein will be used for (determination of specific enzymatic activity, protein crystallization, and interaction studies etc.). Protein stabilizers or serum albumins (e.g. BSA) can be added to the protein to extend the shelf life. The presence of albumin in a sample is not an obstacle when the protein activity is tested in the following steps, but can interfere in structural studies. In contrast, the addition of substances that prevent from the growth of microorganisms is recommended, as they reduce the freezing point, or preserve the protein in the solution. When adding such substances, it is necessary to perform protein stability studies over time. It should be mentioned that there has been no universal stabilizing solution developed for storage of all the various protein samples yet. It is also necessary to be cautious about optimal conditions for the specific protein sample that can vary during storage (Tab. 2) since storage may alter intermolecular interactions. Further, optimal protein storage conditions may not be necessarily the same as the conditions for determining the protein activity. Moreover, additives used to ensure protein stability might act as inhibitors of enzymes. That is the reason why interfering substances should be taken into consideration and must be removed or sufficiently diluted before the use of the protein. Currently the market offer comprises many commercial kits with a wide variety of specific solutions to test protein and storage stability under different conditions. Conclusions In this paper, all the steps and factors have been summarized and briefly discussed which could be encountered during production and purification of a recombinant protein of interest, especially from eukaryotic source, and expressed heterogeneously in prokaryotic production system. It is inevitable to mention that despite the simplicity and overall effectiveness of E. coli production system in combination with various commercially available expression vectors, the final amount and purity of a recombinant protein is insufficient and, many times, contaminated, insoluble or inactive. In many cases, when a soluble protein is inactive or not crystallized after all the separation and purification steps, there is an urgent need to go back to the beginning of the whole protocol, find and solve the origin of a problem and try to optimize the whole process. These findings open a wide range of up-to-date issues to be analysed, as well as possibilities how to improve protein features. However, abovementioned hinders can be overcome by investment of resources into more in- depth studies. Bereitgestellt von Slovenská poľnohospodárska knižnica | Heruntergeladen 27.02.20 14:22 UTC Nova Biotechnol Chim (2017) 16(1): 1-11 9 References Andersen CL, Matthey-Dupraz A, Missiakas D, Raina S (1997) A new Escherichia coli gene, dsbG, encodes a periplasmic protein involved in disulphide bond formation, required for recycling DsbA/DsbB and DsbC redox proteins. Mol. Microbiol. 26: 121-132. 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