LEX5Y Eukaryotic Protein Expression IFTAAG Certified QMS according to DIN EN ISO 9001 Reg. No. IC 03214 034 www.jenabioscience.com • • • Jena Bioscience Jena Bioscience Jena Bioscience GmbH was founded in 1998 by a team of scientists from the Max-Planck-lnstitute for Molecular Physiology in Dortmund. 25+ years of academic know how were condensed into the company in order to develop innovative reagents and technologies for the life science market. Since the start up, the company has evolved into an established global reagent supplier with more than 5500 products on stock and > 3000 customers in 50+ countries. Jena Bioscience serves three major client groups: • Research laboratories at universities, industry, government, hospitals and medical schools • Pharmaceutical industry in the process from lead discovery through to pre-clinical stages ■ Laboratory & diagnostic reagent kit producers and re-sellers Our company premises are located in the city of Jena /Germany with a subsidiary in Teltow, in the vicinity of the German capital Berlin. Jena Bioscience's products include nucleosides, nucleotides and their non-natural analogs, recombinant proteins & protein production systems, reagents for the crystallization of biological macromolecules and tailor-made solutions for molecular biology and biochemistry. In our chemistry division, we have hundreds of natural and modified nucleotides available on stock. In addition, with our pre-made building blocks and in-house expertise we manufacture even the most exotic nucleotide analog from mg to kg scale. In the field of recombinant protein production, Jena Bioscience has developed its proprietary LEXSY technology. LEXSY {Leishmania Expression System) is based on a SI -classified unicellular organism that combines easy handling with a full eukaryotic protein folding and modification machinery including mammalian-like glycosylation. LEXSY is primarily used for the expression of proteins that are expressed at low yields or are inactive in the established systems, and expression levels of up to 500 mg/L of culture were achieved. Imprint Design and Layout by: timespin - Digital Communication GmbH Sophienstrasse 1,07743 Jena www.timespin.de Copyright: Please contact Jena Bioscience if you want to use texts and/or images in any format or media. For the crystallization of biological macromolecules - which is the bottleneck in determining the 3D-structure of most proteins - we offer reagents and tools for crystal screening, crystal optimization and phasing that can reduce the time for obtaining a high resolution protein structure from several years to a few days. Our specialized reagents are complemented with a large selection of products for any molecular biology & biochemistry laboratory such as kits for Standard PCR and Real-Time PCR, fluorescent probes, oligonucleotides, cloning enzymes, mutagenesis technologies, and many more... We combine highest quality standards for all our products (certified according to DIN EN ISO 9001) with individualized customer support. We establish direct lines of communication from clients to our in-house scientists, resulting in productive interactions among people with similar research interests who speak the same language. Furthermore, we offer support programs and attractive discount schemes for young scientists establishing their own labs. If you wish to receive more information, just send an e-mail to info@jenabioscience.com Table of Contents Introduction Overcoming limitations of other expression systems LEXSY Configurations 7 In Vivo LEXSY 7 In Vitro LEXSY 75 Applications and selected examples 18 Solubility and functionality of recombinant proteins 18 Mammalian-type glycosylation 19 Expression of complex oligomeric proteins 20 Expression of recombinant antibodies 20 Structural biology: LEXSY proteins for NMR and X-ray crystallography 20 LEXSY in parasitology 21 References 23 International distributors '4L Introduction & Overcoming limitations of other expression systems Jena Bioscience Introduction The Leishmania expression system LEXSY is the proprietary eukaryotic protein expression platform by Jena Bioscience. LEXSY is based on the protozoan host Leishmania tarentoiae and was designed to combine eukaryotic protein synthesis and modification with simplicity and ease of handling. L. tarentoiae is a robust, unicellular, flagellated eukaryotic organism of circa 5 x 15 urn size (Figure 1). It was isolated from lizards Tarentoiae annuiaris and Tarentoiae mauritanica and has been cultivated in axenic culture over decades. It is not pathogenic to mammalians and is fully approved for use in biosafety level 1 laboratories (S1). Figure 1 Cultured Leishmania tarentoiae cells expressing green fluorescent protein LEXSY and all of its components are commercially available world-wide. Licensing is not required for non-profit research institutions such as universities; however, the use of LEXSY and its components for commercial purposes requires a license from Jena Bioscience. For more information and terms of licensing, please contact expression@jenabioscience.com. Overcoming limitations of other expression systems Prokaryotic expression systems such as £ coli lack essential components for protein folding and modification and are therefore, in most cases not suitable for production of functional proteins of higher organisms. Alternative eukaryotic expression systems based on e.g. mammalian or insect cells however, require long development cycles and deliver low protein yields resulting in costs that are magnitudes above those off. coli-produced proteins. Hence, LEXSY was developed in order to make use of its eukaryotic protein synthesis and folding/modification machinery and its simplicity and ease of handling. PROTEIN SYNTHESIS AND FOLDING/MODIFICATION MACHINERY LEXSY FEATURES: ■ Eukaryotic protein synthesis for correct folding (no inclusion bodies) ■ Full range of Post-Translational Modifications including mammalian-type N-glycosylation, glypiation, phosphorylation, acetylation, prenylation, myristoylation, ADP-ribosylation, proteolytic processing and oligomerisation ■ High expression-success rates with yields of up to 500 mg per litre of culture (Figure 2) 4 80 M_ 20 ■ 40 cytosolic proteins 24 secretory proteins 6 membrane proteins n Positive 0.1...1 1...5 expression S01 25 Total Protein yield (mg per litre of culture) Target protein Size (kDa) Yield (mg/L) Cytoplasmic proteins EGFP 28 300 S0D1 16 30 SPEE 35 30 p85 of PI3 kinase 85 3 smmyHC 154 1 Nuclear proteins T7 RNA Pol 100 1 Secreted proteins MHCII-ß 30 500 CRP 23 44 SAG1&2 15/31 10 Fe fusion 39 10 MDP1 45 6 Laminin 332 420 (150+135+135) 0.5 Membrane proteins EGFP-Rab7 (mb-associatecl) 52 12 PDM9 (Type I) 43 0.5 BkrB2-GST (Type I ITM7) 55 0.1 LEXSY performs mammalian-type glycosylation B o o o o oo o o o o oooooo o o Bacteria Yeast e.g. Pichia O (O) O ■ iO)M Insect cells e.g. Sf9/21 (A) (A) ■ ■ o o 0 1 *3rd **th ^ ^ antenna Mammalian cells O Galactose o Man nose O Fucose A N-acetylneuraminic acid ■ N-acetylglucosamine Polypeptide | partial modification Figure 2 \: Expression of 70 proteins was screened in LEXSY. After cultivation for 2-3 days in suspension culture more than 80% were expressed at > 0.1 mg/L of culture. Table: Typical examples of LEXSY-expressed proteins clustered by type of protein. S0D1 = human Cu/Zn superoxide dismutase; EGFP = enhanced green fluorescent protein of A victoria; SPEE = human spermidine synthetase; p85 = bovine Phosphoinositide 3-Kinase regulatory subunit a; smmyHC = heavy chain of human smooth muscle myosine; T7 RNA Pol. = RNA polymerase of phage T7 supplied with nuclear localization signal; MHC ll-p = human Major Histocompatibility Complex II p subunit (Wienhold et al, not published); CRP = human C-reactive protein of pentaxin family; SAG1/2 = surface antigens of Toxoplasma gondii (Fritsche et al. 2008); Fc fusion = N-terminal fusion of DNA binding domain to human Fc fragmant (Figure 17); MDP1 = human renal dipeptidase 1; Laminin 332 = large heterotrimeric human laminin glycoprotein o3|33y2 (Figure 16, Phan etal. 2009); EGFP-Rab7 = EGFPfusion of Ras-associated small GTP-binding protein Rab7 (membrane associated by prenylation); BrkB2-GST = GST fusion of human bradykinin receptor B2 (7TM transmembrane protein); PDM9 = human transmembrane protein with EGF-like and two follistatin-like domains 2 (type I membrane protein N out). B: Glycosylation in LEXSY was investigated with human erythropoietin (EPO), human interferon gamma (hu IFNy) and host surface glycoprotein GP63. In all cases a biantennary, fully galactosylated, core-a-1,6-fucosylated N-glycan structure was found that is similar to mammalian-type glycosylation (Breitling etal. 2002). Overcoming limitations of other expression systems Jena Bioscience SIMPLICITY AND EASE OF HANDLING - FROM GENE TO PROTEIN WITHIN SIX WEEKS LEXSY exerts ■ Biosafety level 1 (SI, as £ coli) ■ Easy plasmid generation in £ coli shuttle vectors ■ High transfection efficiencies using established electroporation protocols ■ Cultivation in inexpensive media at 26°C - no cell culture equipment necessary ■ Rapid growth of LEXSY expression strains to high cell densities (109 cells/ml) ■ Easy harvest and downstream processing (Figure 3). rnr v — Po - I ta I hyg I ssu ssu } LEXSY host T7-TR Po 1 target marker expression cassette Po target blecherry Figure 5 Architecture of inducible LEXSY. Engineered LEXSY hostT7-TR expresses bacterialT7 RNA polymerase andTET repressor. Following transfection target gene is expressed under control of T7 promoter-TET operator assembly (more details are given in the text). Both, the constitutive and the inducible configuration, permit intracellular or secretory expression of proteins from the same vector simply by choosing the way of cloning: Secretion is achieved by fusion of the mature part of the target gene to a Leishmania signal peptide encoding sequence present on the vector (Figure 6), and is recommended for proteins that undergo Post-Translational Modifications such as disulfide bond formation or glycosylation. LEXSY was shown to yield exceptionally homogeneous mammalian-type N-glycosylation patterns (Breitling et al. 2002, Figures 2 and 14). The first step of the construction of recombinant LEXSY strains - cloning of the target gene into a LEXSY expression vector - is performed in £ coli. Cloning of the target gene into the multiple cloning sites provides essential non-translated flanking regions already optimized for the LEXSY host. All pLEXSY expression vectors bear compatible cloning sites for insertion/ shuffling of expression cassettes and - in addition - a CHexa-Histidine tag for protein detection and affinity purification. Swal Swal 5'odc utr1 ' SP pLEXSY_l-3 He. 8 kbp marker /Bglll ,Ncol /Slal ,Sall -Narl -Xbal MspCI Z-Kpnl « Figure 6 Maps of the integrative pLEXSY vector family for constitutive (left) or inducible (right) expression. 5' and 3'ssu or 5'and 3' ode are regions for homologous recombination into the host chromosome following linearization of the expression plasmid with Swal. Utr1, utr2 and utr3 are optimized non-translated gene-flanking regions providing the splicing signals for posttranscriptional mRNA processing for expression of target and marker genes in the LEXSY host. SP designates the signal peptide of L. mexicana secreted acid phosphatase LMSAP1 (Wiese etal. 1995) and H6 the hexa-Histidine stretch. Alternative cloning strategies result in cytosolic (c) or secretory (s) expression of the target proteins. In the constitutive system sat (streptothricine acetyltransferase), neo (aminoglucoside phosphotransferase), hyg (hygromycin phosphotransferase), or ble (bleomycin resistance) genes are available as selection markers for selection with the antibiotics LEXSY NTC, -Neo, -Hyg, or -Bleo, respectively. In the inducible system blecherry, ble and neo resistance genes are available for selection with LEXSY Bleo, or -Neo. 8 LEXSY Configurations Table 2 In Vivo LEXSY configurations Parameters Constitutive LEXSY Inducible LEXSY LEXSYcon LEXSinduce Intracellular Secretory Intracellular Secretory Typical cultivation time 2-4 days 2-4 days 1-3 days 1-3 days Number of available selection markers 4m 4m jm 2121 [1] 4 alternative selection antibiotics available (LEXSY NTC, LEXSY Hygro, LEXSY Bleo, LEXSY Neo) - Page 13 [2] 2 alternative selection antibiotics available (LEXSY Bleo, LEXSY Neo) Furthermore, the inducible LEXSY is available as integrative or episomal version. In the integrative version the expression cassette is stably integrated into the chromosomal ornithin decarboxylase (ode) locus whereas the episomal version makes use of amplification and oligomerisation of expression plasmids maintained extrachromosomally as self-replicating episomes (Kushnir eta I. 2011). Finally, the inducible configuration pLEXSYJ-blecherry enables efficient screening of high expression clones and online monitoring of induction using Cherry-fluorescence (Figure 7). This was achieved by fusion of the ble resistance and cherry fluorescence genes. Plasmid constructs Transfection Clonal selection & pre-screening by cherry color Expansion T7-Pro- G cue of mote r Interest BleCherry Marker Small-scale cultivation of positive clones Identification of clones with highest expression yields by fluorescence measurement V ■ J= I --*-í l—l 1 '- =. = c 1 Q ■ . "1 Ir- -il !■—.1 Ii- ■« In -n Scale-up cultivation S 9 10 11 12 Large-scale cultivation of highly expressing clones Cell harvest Time of harvest control by fluorescence measurement Purification of protein of interest ;.l Fractions containing protein of interest ^ BleCherry Marker ^ Protein of interest Figure 7 Inducible protein expression with pLEXSYJ-blecherry architecture employing coupled expression of target and BleCherry proteins. Row 1: LEXSY expression plasmid is constructed by insertion of the gene of interest into the pLEXSYJ-blecherry expression vector (Cat. No. EGE-243 or EGE-251). Following transfection of LEXSY host T7-TR, cells are spread onto nitrocellulose membranes covering selective LEXSY BHI Agar and incubated for ca. 7 days at 26°C. After appearance of recombinant colonies the membrane is transferred onto a fresh LEXSY BHI Agar plate containing the inducer tetracycline and incubated for 1-2 additional days. Recombinant clones are pre-screened by cherry color in daylight. Most intense cherry-colored colonies are expanded by small-scale cultivation in suspension for further evaluation. Row 2: Clones with the highest expression yields are identified in multi well plates by fluorescence measurement of samples of the cultures 48 h post induction. Most productive clones are expanded for upscale. Row 3: Large-scale fermentation (10-30 L scale) is performed for purification of large amounts of proteins. The optimal time of harvest is determined by online fluorescence measurements of culture samples. At the optimal time point the cells are harvested for downstream processing. 9 LEXSY Configurations Jena Bioscience IN VIVO LEXSY PRODUCTS For getting started with In Vivo LEXSY, Expression Kits are available that contain all components for construction of expression strains and setup of expression evaluation (Figure 8). expression vector for intracellular or secretory expression, four markers available sequencing and diagnostic primer pairs antibiotic for selection of recombinant strains LEXSY medium all components for 1 litre of growth medium Figure 8 The constitutive LEXSY Expression Kits contain LEXSY host strain L.tarentolae P10, pLEXSY-2 expression vector, all components for preparation of 1 L of medium including selective antibiotic, primer sets for insert sequencing and diagnostic PCR and a detailed and easy-to-follow manual. The inducible LEXSY Expression Kits contain instead LEXSY host strain T7-TR expressing T7 RNA polymerase and TET repressor and the pLEXSYJ vector. In addition, all single components as well as auxiliary products are available separately. The constitutive LEXSY Expression Kits are available with four alternative selection markers (LEXSY NTC, -Neo, -Hyg, or -Bleo selection). The inducible LEXSY Expression Kits are available with three alternative selection marker genes (blecherry, ble, orneo) for selection with the antibiotics LEXSY Bleo, or-Neo. LEXSY EXPRESSION KITS Product Cat. No. Amount Price (EUR) LEXSYcon2 Expression Kit for constitutive protein expression, contains vector pLEXSY-sat2 or pLEXSY-neo2 or pLEXSY-hyg2 or pLEXSY-ble2 EGE-1300sat EGE-1300neo EGE-1300hyg EGE-1300ble 1 Kit 960,- LEXSinduce3 Expression Kit for inducible protein expression from integrative constructs, contains vector pLEXSY_l-blecherry3 or pLEXSY_l-ble3 or pLEXSYJ-neo 3 EGE-1410blecherry EGE-1410ble EGE-1410neo 1 Kit 2.250- LEXSinduce4 Expression Kit for inducible protein expression from episomal constructs, contains vector pLEXSY_IE-blecherry4 EGE-1420blecherry 1 Kit 2.250,- Each Kit contains the expression vector of choice indicated in the product name. The inducible expression kits contain in addition a control vector with the egfp gene inserted into the expression site. For upgrading of the expression kits all LEXSY expression vectors are available also separately. The cloning sites of all pLEXSY vectors are compatible (Figure 6).This enables convenient transfer of target genes between the LEXSY configurations for expression optimization. 10 LEXSY Configurations LEXSY EXPRESSION VECTORS Product pLEXSY-ble2 integrative constitutive expression vector antibiotic selection of transfectants with LEXSY Bleo pLEXSY-hyg2 integrative constitutive expression vector antibiotic selection of transfectants with LEXSY Hygro pLEXSY-neo2 integrative constitutive expression vector antibiotic selection of transfectants with LEXSY Neo pLEXSY-sat2 integrative constitutive expression vector antibiotic selection of transfectants with LEXSY NTC pLEXSY_l-blecherry3 integrative inducible expression vector antibiotic selection of transfectants with LEXSY Bleo, expression monitoring with Cherry fluorescence pLEXSY_l-ble3 integrative inducible expression vector antibiotic selection of transfectants with LEXSY Bleo pLEXSY_l-neo 3 integrative inducible expression vector antibiotic selection of transfectants with LEXSY Neo pLEXSY_IE-blecherry4 episomal inducible expression vector antibiotic selection of transfectants with LEXSY Bleo, expression monitoring with Cherry fluorescence Cat. No. EGE-231 EGE-232 EGE-233 EGE-234 EGE-243 EGE-244 EGE-245 EGE-255 Amount 5 ug 5 ug 5 ug 5 ug 5 ug 5 ug 5 ug 5 ug Price (EUR) 480- 480-480-480- 480- 480- 480- 480- LEXSY CONTROL VECTORS Product pLEXSY-egfp-sat2 integrative constitutive control vector with egfp gene antibiotic selection of transfectants with LEXSY NTC, expression monitoring with EGFP fluorescence pLEXSY-cherry-sat2 integrative constitutive control vector with cherry gene antibiotic selection of transfectants with LEXSY NTC, expression monitoring with Cherry fluorescence pLEXSY-red-sat2 integrative constitutive control vector with Ds red gene antibiotic selection of transfectants with LEXSY NTC, expression monitoring with Ds red fluorescence pLEXSY_l-egfp-blecherry3 integrative inducible control vector with egfp gene antibiotic selection of transfectants with LEXSY Bleo, expression monitoring with EGFP and Cherry fluorescence pLEXSY_l-egfp-ble3 integrative inducible control vector with egfp gene antibiotic selection of transfectants with LEXSY Bleo, expression monitoring with EGFP fluorescence pLEXSY_l-egfp-neo3 integrative inducible control vector with egfp gene antibiotic selection of transfectants with LEXSY Neo, expression monitoring with EGFP fluorescence Cat. No. EGE-235 EGE-236 EGE-237 EGE-246 EGE-247 EGE-248 Amount 5 ug 5 ug 5 ug 5 ug 5 ug 5 ug Price (EUR) 480,- 480- 480- 480,- 480,- 480- LEXSY Configurations Jena Bioscience Once constructed, the expression plasmids are used for transfection of the LEXSY host strains by electroporation. Following electroporation, recombinant strains are selected either polyclonally or clonally. Polyclonal selection results from addition of selection antibiotic to the cell suspension whereas clonal selection is achieved by plating of cells onto solid selection media. For convenience LEXSY Plating Kits were developed that contain all components required to set up clonal selection. The three different kit formats differ by auxiliary components dependent on preferences of customer laboratories. LEXSY PLATING KITS Product Cat. No. Amount Price (EUR) LEXSY Plating Kit comfort components for solid medium, LEXSY BHI- and fetal-calf-serum-based, with nitrocellulose membranes, spatula, dishes & serological pipettes /1L-451 for 40 plates 300,- LEXSY Plating Kit core components for solid medium, LEXSY BHI- and fetal-calf-serum-based, without nitrocellulose membranes, spatula, dishes & serological pipettes /lL-452 for 40 plates 650- LEXSY Plating Kit basic components for solid medium, LEXSY BHI-based, without fetal-calf-serum, nitrocellulose membranes, spatula, dishes & serological pipettes /lL-453 for 40 plates 630- The plating kits provide components proven to ensure high efficiency of colony formation following plating of transfected LEXSY cells. Figure 9 shows typical plating efficiencies over a broad range of transforming DNA concentrations. Figure 9 LEXSY plating efficiencies for an integrative construct. LEXSY host was cultivated at 26°C in suspension culture in LEXSY BHI Medium and electroporated 24 h post inoculation at cell density of 6 x 107 cells/ml by low voltage procedure with settings 450 V and 450 uF. Linearized DNA was added at concentrations indicated in the table. Three aliquots of 2 ml were withdrawn from each o/n culture of electroporated cells, sedimented and spread onto LEXSY BHI Agar. Colonies were counted 7 d post plating. For details of the protocol refer to LEXSY manuals. 12 LEXSY Configurations Selection of recombinant LEXSY strains is performed with antibiotics used also for other eukaryotic hosts. Jena Bioscience offers LEXSY Selection Antibiotics which are evaluated for efficient selection of recombinant LEXSY strains and are provided as powder as well as sterile ready to go 1.000 x stock solutions. LEXSY SELECTION ANTIBIOTICS Product Cat. No. Amount Price (EUR) AB-101S 1 ml 40- Nourseothricin (NTC) AB-101L 5 ml 160- sterile ready to go 1.000x stock solution, 100 mg/ml AB-101-10ML 10ml 300- AB-101-50ML 50 ml 1.200- AB-102L ig 178- Nourseothricin (NTC) AB-102XL 5g 870- powder (non-sterile) AB-102-25G 25 g 3.525 - AB-102-100G 100 g 12.350- LEXSY Bleo AB103S 1 ml 80- sterile ready to go 1.000x stock solution, 100 mg/ml AB-103L 5 ml 320- LEXSY Hygro AB-104S 1 ml 80- sterile ready to go 1.000x stock solution, 100 mg/ml AB-104L 5 ml 320- LEXSY Neo AB-105S 1 ml 40- sterile ready to go 1.000x stock solution, 50 mg/ml AB-105L 5 ml 160- For optimal selection LEXSY NTC, LEXSY Bleo and LEXSY Hygro are used at 100 ug/ml and LEXSY Neo at 50 ug/ml final concentrations in the cultivation medium. 13 LEXSY Configurations Jena Bioscience LEXSY suspension cultures are grown in complex or in synthetic media. For routine cultivations, transfection, cryoconservation and expression evaluation Complex LEXSY BHI Cultivation Media are used, based on brain and heart extracts. For cultivation in animal-free media Jena Bioscience offers LEXSY YS media based on yeast and soy extracts. In both types of complex media cell densities up to 5x 108 cells/ml are reached in agitated laboratory cultivations. COMPLEX LEXSY CULTIVATION MEDIA Product Cat. No. Amount Price (EUR) LEXSY BHI - Liquid Media Kit sterile, brain-heart-infusion based medium, recommended for strain maintenance, electroporation, expression evaluation and cryoconservation ML-411S 1 L 90- ML-411L 5 L 360- LEXSY BHI - Powder Media Kit Brain-heart-infusion based medium, recommended for strain ML-412S fori L 45- ML-412L for 5 L 180- maintenance, electroporation, expression evaluation and cryoconservation ML-412XL for 10 L 350- ML-412XXL for 50 L 1400- LEXSY YS- Liquid Media Kit ML-431S 1 L 100- sterile, yeast-soybean based, casein-free medium medium free of animal components ML-431L 5L 400- ML-432S fori L 50- LEXSY YS- Powder Media Kit yeast-soybean based, casein-free for medium free of animal components ML-432L for 5 L 200- ML-432XL for 10 L 375 - ML-432XXL for 50 L 1500- If cultivation of LEXSY strains is required in protein-free defined media, Synthetic LEXSY Cultivation Media are available. They are optimized for high cell densities up to 3x108 cells/ml in agitated laboratory cultivations. SYNTHETIC LEXSY CULTIVATION MEDIA Product Cat. No. Amount Price (EUR) Synthetic LEXSY Medium - liquid, ready-to-grow, ML-103S 1 L 150- sterile, contains Hemin and Pen-Strep, shelf life 2 weeks ML-103L 5 L 600- Synthetic LEXSY - Liquid Media Kit ML-107S 1 L 150- sterile, with Hemin and Pen-Strep stock solutions, shelf life 6 months ML-107L 5L 600- I LEXSY Cultivation Media Kits contain ready-to-go stock solutions of Hemin and PenStrep, which must be added before use. These additives are also available separately. © 14 LEXSY Configurations ADDITIVES FOR LEXSY CULTIVATION MEDIA Product Cat. No. Amount Price (EUR) ML-108S 2 ml (for 1 L) 10- Hemin (porcine) ML-108L 10 ml (for 5 L) 40- sterile 500x stock solution in 50% triethanolamine ML-108XL 20 ml (for 10 L) 75- ML-108XXL 100 ml (for 50 L) 300- ML-105S 5 ml (for 1 L) 10- Pen-Strep ML-105L 25 ml (for 5 L) 40- sterile 200x stock solution of penicillin and streptomycin ML-105XL 50 ml (for 10 L) 75- ML-105XXL 250 ml (for 50 L) 300- Hemin is essential for growth of LEXSY cultures. Addition of Pen-Strep prevents potential bacterial contaminations. Following addition of these components the media are stable for two weeks. If the completed media are to be used after this period, appropriate amounts of additives have to be re-added. In Vitro LEXSY Cell-free expression has become a powerful method for production of recombinant proteins and plays a central role in a wide variety of applications such as functional analysis and biochemical characterization of proteins and protein interactions, investigation of protein translation mechanisms, protein engineering, in vitro evolution, and structural biology (Katzen etal. 2005). Its multiplexed format can be used for production of protein arrays for drug screening and diagnostics (He etal. 2007). The main advantages of cell-free protein expression are its rapidity of only few hours and its independence of living host-organisms. These features enable very fast generation of results and greatly alleviate typical in vivo expression problems caused by toxicity and/or degradation of the protein of interest. Our In Vitro LEXSY Translation System is a NEW, rapid, convenient, flexible and cost-efficient tool for production of recombinant proteins from DNA templates in a single-tube reaction based on the cell extract of the protozoon Leishmania tarentolae (Mureev etal. 2009, Kovtun etal. 2010 & 2011). In contrast to £ coli in vitro translation, LEXSY contains chaperones for correct folding of proteins of higher eukaryotes (Kovtun et a I. 2010). Further, compared to insect, rabbit and wheat germ systems, LEXSY yielded significant higher expression levels (Figure 10). Finally, our In Vitro LEXSY Translation System allows efficient suppression of background translation that is often required in other cell-free systems. A simple anti-splice leader oligonucleotide blocks translation of endogenous mRNA. Figure 10 Coupled transcription-translation of PCR generated EGFP template in LEXSY compared to other commercially available in vitro translation systems. The EGFP ORF was amplified by overlap-extension PCR and fused individually with the translational leaders according to the instructions of the cell-free systems manufactures. For details refer to Mureev etal. 2009. Rabbit Insect Wheat Germ 120 140 Time (min) 15 LEXSY Configurations Jena Bioscience Dependent on the wayoftemplate preparation two principle versionsof/n Wfro LEXSY are available, that are plasmid based or PCR based versions (Figure 11). The Plasmid-based In Vitro LEXSY Translation is recommended for high-yield and/or large volume reactions. It is also recommended for open reading frames larger than 2500 bp and requires sub-cloning of the target ORF into the pLEXSYJnvitro vector. The PCR-based In Vitro LEXSY Translation is rapid and flexible. It utilizes PCR-mediated fusion of the target ORF to a T7 promoter and leader sequence by overlap extension (OE-PCR) technique and does not require any cloning step. Therefore, this approach allows rapid generation of large protein libraries directly from unpurified PCR products. Plasmid-based In Vitro LEXSY -for high yields- Target ORF PCR-based In Vitro LEXSY - for high throughput - Ncol Bglll Xhol 17 Po leader adaptor ■ SITS OE PCR T7 Po leader adaptor ORF Template generation by cloning of target genes into plasmid Template generation by direct amplification of target DNA with overlap extension (OE) PCR Cell-free production of proteins in LEXSY eel I extracts 1 Z3MW4 567 Plasmid-based PCR-based Cell-free production of EGFP reference protein with plasmid-based (lanes 1-3) and PCR-based (lanes 4-6) In Vitro LEXSY. Lane 7: negative control without template. MW: molecular size marker. The in vitro reactions were carried out for 2 h at 20°C, resolved on 12% SDS-PAGE and in situ visualized on a UVtransillumi-nator. Figure 11 Flow chart of the two principle versions of in Vitro LEXSY. DNA templates for in vitro translation can be generated either by cloning into pLEXSY_ in vitro vector (A) or by a two step-PCR amplification procedure (B). The DNA templates are transcribed and translated in a single-tube reaction with LEXSY cell extracts (C). The in vitro produced proteins can be detected by fluorescence scanning in case of EGFP fusion proteins , by Western blotting or other techniques. Abbrevations: T7Po = T7 RNA polymerase promoter, MCS1 & MCS2 = multiple cloning sites for replacement of vector-borne EGFP control gene and for N'or C'in-frame fusions to the EGFP gene resp., Leader = 63 nt poly-TTTTA sequence for generation of unstructured 5'end of template mRNA, Adaptor = 24 nt DNA sequence (encoding KDIKHVSE peptide) for overlap extension PCR (OE PCR), MYC = Myc-tag, to =T7 transcription terminator, bla = ampicillin resistance gene, ori E.c. = replication origin for Escherichia coii, SITS = species independent translation sequence consisting of T7Po + leader- + adaptor sequences. For more details, refer to the in Vitro LEXSY manuals. 16 www.jenabioscience.com LEXSY Configurations (® IN VITRO LEXSY PRODUCTS Product Cat. No. Amount Price (EUR) In Vitro LEXSY Translation Kit 15 reactions for plasmid based cell-free protein synthesis EGE-2002-15 1 Kit 225- In Vitro LEXSY Translation Kit 15 reactions for PCR based cell-free protein synthesis EGE-2010-15 1 Kit 225- In Vitro LEXSY Translation Cell Extract 15 reactions for cell-free protein synthesis EGE-260 250 ul 150- Figure 12 shows examples of proteins produced with In Vitro LEXSY Translation Kit. Enhanced Green Fluorescent Protein (EGFP) and its fusion proteins can conveniently be detected directly in SDS-PAGE gels by in situ fluorescence scanning (A) or isolated by affinity chromatography on a GFP binding matrix for subsequent detection by conventional Coomassie staining (B). For non-fluorescent protein targets Western blotting can be used for visualization. # # £ c2S 120-86-47- 34" 26- 20- Rab7-EGFP SOD-EGFP GST-EGFP EGFP B 2 3 4 5 6 7 MW Figure 12 Cell-free expression of EGFP fusion proteins with In Vitro LEXSY. i: Simultaneous in vitro co-expression of four proteins in a single extract (EGFP and three EGFP fusion proteins, Rab7 = Ras-related small GTPase7, SOD = Cu/Zn superoxide dismutase, GST = Glutathione-S-Transferase). The in vitro reactions were resolved on SDS-PAGE and the products detected by in situ fluorescence (Adapted from Mureev et al. 2009). All proteins are present at similar yields indicating suitability of the system for production of heteromeric protein complexes. 5: Purification of in vitro produced EGFP fusion proteins. 1=Rab8 (Ras-related small GTPase8)-EGFP, 2 = Cog5 (Complex of Golgi5)-EGFP, 3 =Cog8 (Complex of Golgi8)-EGFP, 4 = Rab1 ( Ras-related small GTPase1)-EGFP, 5 = RabGGTB (Geranyl-Geranyl Transferase B)-EGFP, 6 = MBP (Maltose Binding Protein)-EGFR 1 = EGFP In vitro reactions and GFP matrix purification were performed as described in the In Vitro LEXSY user manual. The purified target proteins were resolved by SDS-PAGE and Coomassie stained. Right lane, molecular size protein marker (kDa). Adapted from Kovtun etal. 2010. 17 Applications and selected examples Applications and selected examples Solubility and functionality of recombinant proteins Incorrect folding and insufficient solubility - resulting in compromised biological activity - are the major shortcomings of prokaryotic protein production systems (Zerbs et al. 2009, Makrides 1996). Due to LEXSY's fully eukaryotic protein synthesis/folding/modification machinery most proteins of higher organisms expressed in LEXSY are correctly folded and processed and therefore, are obtained in a fully functional state (Table 3). Table 3 Selected examples of LEXSY-expressed proteins with full biologic activity Protein Localisation Origin Reference Erythropoietin secreted human Breitling etal. 2002 Surface Antigen 1 & 2 secreted Toxoplasma gondii Ebert etal. 2007 not publ. Proprotein Convertase 4 secreted rat Basakefo/. 2008 Laminin-332 secreted human Phanefo/. 2009 Cu/Zn superoxide dismutase cytosolic human Gazdag etal. 2010 Tissue Plasminogen Activator secreted human Hemayatkarefo/. 2010 N-Acetyl Serotonin Methyl Transferase (ASMT) cytosolic human Ben-Abdallah etal. 2010 Hydroxynitrile Lyase (MeHNL) cytosolic cassava plant Dadashipourefal. 2011 Coagulation factorVII cytosolic human Mirzaahmadiefa/2011 Proprotein Convertase 4 (PC4) is a Ca++ dependent mammalian subtilase (proprotein convertase subtilisin kexin PCSK), which plays a key role in fertilization. Recombinant PC4 could previously be generated only in extremely poor yields using rat GH4C1 or insect Hi5 cells. Using LEXSY, full length and truncated forms of this enzyme were expressed, and soluble, active protein was purified in high yields. Biochemical analysis demonstrated high specific activity, which was superior to PC4 obtained from GH4C1 or Hi5 cells. The substrate specificity found confirmed its biological role and allowed inhibitor design for therapeutic and clinical applications (Basakefo/. 2008). Tissue Plasminogen activator (t-PA) is a serine protease with 17 disulfide bonds that need to be correctly formed for the enzyme's biological activity. Different expression systems (yeast, insect cells, transgeneic plants) have been tried for production of recombinant human t-PA but yielded unsatisfactory results due to poor secretion , improper folding and hyper-glycosylation. At present, human t-PA is mainly produced at large scale in Chinese hamster ovary (CHO) cells, however, uncontrollable variability in mammalian cell culture processes make development of expressing cell lines laborious and time-consuming. Moreover, high costs of cell culture media and contamination with viruses and prions are additional problems associated with the use of mammalian cells. LEXSY in contrast alleviates these problems and yielded correctly folded t-PA with full biological active (Hemayatkar etal. 2010). N-Acetyl Serotonin Methyl Transferase (ASMT) is the last enzyme in the melatonin synthesis pathway and possibly involved in autism-related disorders. Attempts to produce human ASMT in recombinant E.coliyielded only insoluble and heavily degraded material. In contrast, recombinant ASMT was produced in soluble, active form and purified in milligram amounts when expressed in LEXSY (Ben-Abdallah etal. 2010). Hydroxynitrile lyase from cassava plant Manihot esculenta (MeHNL) is involved in cyanogenesis in this higher plant. Dadashipour et al. (2011) compared expression and features of MeHNL in E.coli, Pichia pastoris, Leishmania tarentolae and two cell-free translation systems. While the wild type enzyme formed inclusion bodies when expressed in £ coli it could be expressed in soluble form in L. tarentolae and Pichia pastoris. Moreover, the wild-type and mutant enzyme showed high activity for both proteins (up to 10 U/ml) in the eukaryotic host L. tarentolae and Pichia pastoris, while those off. coli exhibited about 1 and 15 U/ml, respectively. 18 Mammalian-type glycosylation Glycosylation is a major posttranslational modification of a large variety of secreted and membrane proteins. It occurs in more than 50% of all human proteins (Rich et al. 2009) and is often a pivotal factor for folding, function and stability. Glycoproteins account for about 60% of the therapeutic protein market with annual growth rates of >20% (Gerngross 2004). Due to the absence of glycosylation pathways in prokaryotes, recombinant glycosylated proteins cannot be produced in e.g. bacteria. Further, glycosylation in most alternative eukaryotic expression hosts such as yeast and insect differs largely from the desired mammalian-type glycosylation (Figure 2B). Despite several improvements including glycoengineering have been reported for these two systems, an expression system with adequate mammalian-type glycosylation is still highly desirable for protein expression in research, diagnostics and pharmaceutical applications. A CHO LEXSY Glycosylation in LEXSY was thoroughly investigated using recombinant human erythropoietin (EPO) as a model. EPO expressed in LEXSY was shown to be efficiently secreted into the culture medium, natively processed at the N-terminus and fully biologically active. Glycosylation analysis revealed two glycans, a complex mammalian-type biantennary oligosaccharide and the Man3GlcNAc2 core structure (Figure 13). LEXSY is thus the first biotechnologically useful unicellular eukaryotic system producing biantennary fully galactosylated, core-a-1,6-fucosylated N-glycans. In addition, the N-glycosylation pattern was exceptionally homogenous consisting of only two defined glycoforms, while glycoproteins from other eukaryotes are typically heterogenous multi-glycoform populations (Figure 13 A & B). LEXSY-derived homogenous protein preparations are therefore expected to be prone to crystallization and subsequent structure determination. o oo a 32S§°--£ CHO Man al 6 Man ßl -» 4 GlcNAc ßl -» 4 GlcNAc Man al Gal ßl ■» 4 GlcNAc ßl ■» 2 Man a1 6 Man ßl -» 4 GlcNAc ßl -» 4 GlcNAc 3 7J Gal ßl ■» 4 GlcNAc ßl ■» 2 Man al Figure 13 Analysis of recombinant human erythropoietin isolated from culture supernatants of a LEXSY expression strain. Western blot of recombinant human EPO produced in CHO cells (1) and LEXSY secreted EPO before (2) and after (3) de-glycosylation with N-glycosidase F (PNG). !: Electrophoretic resolution of heterogenous population of CHO-derived EPO. Glycan structures are depicted at the left. '.: Enzymatic resolution of complex and core glycan structures released from LEXSY-produced EPO (for details refer to Breitling et al. 2002). Similar glycosylation profiles were also found in other LEXSY-produced proteins including human interferon-y (IFN-y) and host major surface protein GP63 suggesting this to be a common feature of all recombinant glycoproteins produced in LEXSY. 19 Applications and selected examples Jena Bioscience Expression of complex oligomeric proteins Many proteins of higher organisms are oligomers consisting of more than one polypeptide chain, and recombinant production of these complexes in an active form often requires simultaneous co-expression of the individual polypeptides. LEXSY allows up to four different antibiotic selection markers to be used for expression of up to four different proteins simultaneously facilitating production of functional oligomers. For example, LEXSY was employed to express human laminin-332 (a3p3y2), a large heterotrimeric glycoprotein and essential component of epithelial basal lamina that promotes cell adhesion and migration (Phan etal. 2009) (Figure 14). Alternatively, avoiding limitation by availability of selection markers in vivo, oligomeric proteins can be obtained using In Vitro LEXSY by co-expession of the respective polypeptides in the same extract (Figure 12A). 1 2 3 Figure 14 Model of heterotrimeric laminin-332 (left) and Western blot of purified 420 kDa laminin heterotrimer separated under non-reducing conditions. Lanes 1 molecular size marker (kDa), 2 laminin from 293-F cells (2 forms), 3 laminin from LEXSY (one defined form) after Phan et al. (2009). Expression of recombinant antibodies Recombinant production of antibodies with focus on monoclonal antibodies (MAbs) has become a challenging task due to the rapidly expanding pharmaceutical and diagnostic markets. Currently more than 20 MAbs are clinically approved and ca. 300 MAbs are under development in Clinical Phases l-lll.The annual demand of the leading 9 MAbs was estimated to be more than 2.200 kg per year (Werner 2011). LEXSY was evaluated for production of heavy and light chains of human IgG, single chain antibodies and Fc fusions. Recombinant Fc fusions were efficiently expressed in LEXSY, completely secreted to the culture medium and one-step affinity purified with Protein A sepharose with yields of ca. 10 mg/L. SDS PAGE analysis demonstrated that the proteins were secreted in the native configuration as dimers (Figure 15). Figure 15 Purification of Fc fusion protein from LEXSY cultivation medium. Lane 1 molecular size marker, 2-3 Protein A sepharose-purified Fc fusion under reducing conditions, 4 dto. non-reducing conditions (JBS not published). Structural biology: LEXSY proteins for NMR and X-ray crystallography The applicability of LEXSY for structural biology was demonstrated by successful 15N-HSQC NMR analysis of a 28 kDa 15N-Val labeled protein purified from recombinant LEXSY strain grown in a synthetic LEXSY cultivation medium (Figure 16). All 18 Val residues of the in vivo labeled protein could be completely assigned in 15N-HSQC NMR spectrum in full agreement with X-ray crystallography (Niculae etal. 2006). Since Leishmania tarentolae is auxotrophic for 11 amino acids and can be grown in chemically defined media, multiple options for labeling strategies are available. Alternatively to chemically defined media labeling strategies in complex media were developed (Foldynova-Trantirkova etal. 2009). random colls a-hellcBB i loops 1 V.6 ÄÖ SS SO 7Jä tr'Jr OJO Figure 16 15N-HSQCNMRanalysisof15N-Val labeled EGFP purified from recombinant LEXSY strain. For detailed description refer to Niculae et a/. (2006). 20 It was shown that LEXSY-expressed proteins can be subjected successfully to crystallography and X-ray analysis. The resolution of a new protein structure was achieved for LEXSY expressed hu Cu/Zn superoxide dismutase SOD1 (Figure 17). In addition, the exceptionally homogeneous glycosylate pattern of LEXSY-produced proteins can be a remarkable advantage for structural analysis of glycoproteins (see also chapter Mammalian-type glycosylation). Figure 17 Structure determination of the new P2 2 2 crystal form of LEXSY-produced human Cu/Zn superoxide dismutase (SOD1). The asymmetric unit contains six SOD dimers arranged as two triangular wheels around sulfate ions. The wheels are arranged in a side-to-side fashion (Gazdag etal. 2010). LEXSY in parasitology Leishmania tarentolaeh a close relative to pathogenic Leishmania species as well as to other pathogens such asTrypanosomes, Piasmodium and Toxopiasma (Figure 18). Due to this evolutionary proximity, the LEXSY technology is efficiently expressing parasite proteins with ■ High yields ■ Correct protein folding ■ Native post-translational modifications Figure 18 Leishmania tarentoiae-based LEXSY is evolutionary closely related to a number of the most common parasites and was used for overexpression and purification of functional parasite proteins. Western blot of 93 kDa J-binding protein (JBP1) of Leishmania sp. Lane 1 = host control, lane 2 = induced culture, lane 3 = non-induced culture (Courtesy of S. Vainio,The Netherlands Cancer Institute, Amsterdam). B: Coomassie stain of immunoreactive surface proteins SAG1 (28 kDa) and SAG2 (15 kDa) of Toxopiasma gondii. Lanes 1 and 5 = host controls, lanes 2 and 3 = SAG1, lane 4 = SAG 2 secreted to the culture medium (Courtesy of M. Ebert, FZMB, Erfurt). For more examples, refer to figure 19 A & D. 21 Applications and selected examples Jena Bioscience In addition, the expression vectors developed for LEXSYcan be used for creation of transgenic strains of other Leishmania species including L. amazonensis, L. donovani, L. infantum, L. major, L. mexicana and also Crithidia sp. as well as the plant parasite Phytomonas serpens (Figure 19B-C). These features of LEXSY enable functional characterization of parasite proteins, investigation of parasite-host interactions, in vivo and in vitro screening of anti-leishmanial drugs and vaccine development. Figure 19 V: Expression and functional analysis of the catalytic domain of ci-N-acetylglucosaminyltransferase from Trypanosoma cruzi (TcOGNT2cat) in LEXSY by Western blotting (left) and enzymatic activities (right). P10 = non-transfected host strain; wt = P10 expressing wild-type TcOGNT2cat; D234A and D234N = single point mutants (from Heise etal. 2009). Subcellular localization of ferrous iron transporter LIT1 expressed in L. amazonensis Alitl promastigotes using pLEXSY constructs. Immunofluorescence demonstrated different targeting of wild type and mutant proteins to the plasma membrane. LIT1 immunofluorescence = green, parasite DNA = blue, FITC = anti-LITI IF on fixed/non-permeabilized promastigotes, FITC-LIVE = anti-LITI IF on live promastigotes (from Jacques etal. 2010). '.: EGFP imaging in L. major reporter strain stably transfected with pLEXSY-egfp construct by Epi-fluorescence microscopy of recombinant L. major promastigotes (left) and intracellular amastigotes in bone marrow-derived macrophages (right) (from Bolhassani etal. 2011). 9: Expression of protozoon RabGTPases originating from L. tarentolae or P. falciparum in PCR-based In Vitro LEXSY. Coomassie stained SDS-PAGE gel loaded with EGFP-Rab GTPases eluted from a GFP binding matrix. For details see In Vitro LEXSY manual (adapted from Kovtun etal. 2010). 22 References Basak etal. (2008) Recombinant proprotein convertase 4 (PC4) from Leishmania tarentolae expression system: Purification, biochemical study and inhibitor design. Protein Expression and Purification 60:117. Ben-Abdallah etal. (2010) Production of soluble, active acetyl serotonin methyl transferase in Leishmania tarentolae. Protein Expression and Purification in press 75:114. Bolhassani etal. (2011) Fluorescent Leishmania species: Development of stable GFP expression and its application for in vitro and in vivo studies. Experimental Parasitology127':637. Breitling etal. (2002) Non-pathogenic trypanosomatid protozoa as a platform for protein research and production. Protein Expression and Purification 25:209. Dadashipour etal. (2011) Comparative expression of wild-type and highly soluble mutant His103Leu of hydroxynitrile lyase from Manihot esculenta in prokaryotic and eukaryotic expression systems. 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Gerngross (2004) Advances in the production of human therapeutic proteins in yeasts and filamentous fungi. Nature Biotechnology 22:1409. He etal. (2007) Arraying proteins by cell-free synthesis. BiomoecularEngineering 24:375-380. Heise et al. (2009) Molecular analysis of a UDP-GlcNAcpolypeptide a-N-acetylglucosaminyl-transferase implicated in the initiation of mucin-type O-glycosylation in Trypanosoma cruzi. Glycobiology 19:918. Hemayatkar et al. (2010) Increased expression of recombinant human tissue plasminogen activator in Leishmania tarentolae. Biotechnology Journal 5:1198. Jacques et al. (2010) Functional characterization of LIT1, the Leishmania amazonensis ferrous iron transporter. Molecular & Biochemical Parasitology 170:28. Katzen etal. (2005) The past, present and future of cell-free protein synthesis. Trends in Biotechnology 23:150-156. Kovtun etal. (2010) Towards the Construction of Expressed Proteomes Using a Leishmania tarentolae Based Cell-Free Expression System. PLOSone 5:e14388. Kovtun etal. (2011) Leishmania cell-free protein expression System. Methods 55:58. Kushnir etal. (2005) Development of an inducible protein expression system based on the protozoan host Leishmania tarentolae. Protein Expression and Purification 42:37. Kushnir etal. (2011) Artificial linear episome-based protein expression system for Protozoon Leishmania tarentolae. Molecular & Biochemical Parasitology 176:69. Lukes et al. (2006) Translational initiation in Leishmania tarentolae and Phytomonas serpens (Kinetoplastida) is strongly influenced by pre-ATG triplet and its 5' sequence context. Molecular & Biochemical Parasitology 148:125. Makrides (1996) Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol. Rev. 60:512. Mirzaahmadiefa/. (2011) Expression of Recombinant Human Coagulation Factor VII by the Lizard Leishmania Expression System. Journal of Biomedicine and Biotechnology doi:10.1155/2011/873874. Mureev etal. (2009) Species-independent translational leaders facilitate cell-free expression. Nature Biotechnology 27:747. Niculae etal. (2006) Isotopic labeling of recombinant proteins expressed in the protozoan host Leishmania tarentolae. Protein Expression and Purification 48:167. Phan et al. (2009) The production of recombinant human laminin-332 in a Leishmania tarentolae expression system. Protein Expression and Purification 68:79. Rich etal. (2009) Emerging methods for the production of homogeneous human glycoproteins. Nature Chemical Biology. 5:206. Sodoyer (2004) Expression Systems for the Production of Recombinant Pharmaceuticals. Biodrugs 18:51. Soleimani et al. (2007) Expression of human tissue-type plasminogen activator (t-PA) in Leishmania tarentolae. Biotechnology & Applied Biochemistry 48:55. Werner (2011) Monoclonal Antibodies versus Antibody Fragments/Protein Scaffolds. 4th Halle Conference on Recombinant Protein Production, February 24th-26th, 2011 Halle (Saale). Wiese et al. (1995) Ser/Thr-rich repetitive motifs as targets for phosphoglycan modifications in Leishmania mexicana secreted acid phosphatase. EMBO Journal 14:1067. Zerbs et al. (2009) Bacterial systems for production of heterologous proteins. Methods in Enzymology 463:149. 23 International distributors BioScientific Fly. Ltd. PO Box 78 Gymea NSW 2227 Australia Tel.: 1300 BIOSCI (264724) E-Mail: info@biosci.com.au www.biosci.com.au Sapphire Bioscience Pty Ltd. 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