J. Biochem. 109, 632-637 (1991)

Expression of cDNA for , a -Like Snake

Masahiro Maeda,* Susumu Satoh,* Shingo Suzuki,* Mineo Niwa,*,1 Nobuyuki Itoh,** and Ikuo Yamashina*** *Product Development Laboratories , Fujisawa Pharmaceutical Co., Ltd., Yodogawa-ku, Osaka, Osaka 532; **Department of Biological Chemistry , Faculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, Kyoto 606; and ***Department of Biotechnology, Faculty of Engineering, Kyoto Sangyo University, Kita-ku, Kyoto, Kyoto 603

Received for publication, November 7, 1990

The cloned cDNA for batroxobin has been expressed in E. coli. Batroxobin could only be obtained as intracellular aggregates of fusion , fused with a small peptide. To obtain the mature batroxobin, the recognition sequence for thrombin was inserted between the peptide and the mature batroxobin. This fusion accumulated in an insoluble form and could easily be purified. After site-specific cleavage of the with thrombin, recombinant batroxobin was isolated by preparative electrophoresis. Batrox obin with enzymatic activity was obtained by the refolding of recombinant batroxobin.

Batroxobin [EC 3.4.21.29] is a thrombin-like enzyme duced as cytoplasmic inclusion bodies in E. coli are biologi obtained from , moojeni venom. The molecu cally inactive when the heterogeneous are highly lar weight of batroxobin containing six disulfide bonds was expressed, and such inclusion bodies become soluble only shown to be 29,100. In contrast to thrombin, which con after treatments under denaturing conditions (5). There verts into by removing fibrinopeptides A fore the refolding of the recombinant proteins is necessary and B, batroxobin removes only fibrinopeptide A. The to obtain the native protein. However, in cases of proteins enzyme is currently used clinically for the treatment of containing many disulfide bonds the refolding is often of low thrombotic diseases, since parenteral administration of the efficiency due to inappropriate disulfide bond formation and enzyme causes the conversion of fibrinogen into a fibrin the final products often show low specific activities. derivative which is rapidly degraded through a secondary In this paper, we describe the expression of batroxobin fibrinolytic process and then eliminated via the (1, 2). cDNA, the selective cleavage of the fusion protein with Batroxobin cDNA and have been isolated from a B. thrombin, and successful refolding to obtain biologically atrox, moojeni venom gland cDNA library and the genomic active batroxobin. library, respectively. The sequence deduced from the nucleotide sequence of batroxobin cDNA exhibit MATERIALS AND METHODS ed significant homology with those of eukaryotic serine , indicating that batroxobin is a member of the Materials-Restriction endonucleases were purchased serine family (3). The exon/intron organization of from Takara Shuzo (Kyoto), Toyobo (Osaka), New England the batroxobin gene was different from that of the pro Biolabs (Beverly), and Boehringer Mannheim (Mannheim). thrombin gene but very similar to those of the and T4 DNA and T4 polynucleotide kinase were from genes. These results indicate that batroxobin is Takara Shuzo. DNA polymerase I (Klenow fragment) was not a member of the prothrombin family but is one of the from BRL (Gaithersburg). Thrombin was from Mochida trypsin/kallikrein family. Since the gland is (Tokyo). assumed to have originated from the submaxillary gland, it Strains and Plasmids-E. coli HB101-16 (6) [F-, is conceivable that batroxobin belongs to the glandular recA13, ara-14, proA2, lacY1, galK2, rpsL20 (Smr), kallikrein family (4). xyl-5, mlt-1, hsdS20 (rs-, mg-), supE44, ƒÉ-, TnlO:: Although batroxobin can be purified from venom gland of htpR16] was used as the host for the expression of genes. the snake B. atrox, moojeni, it is difficult to obtain large Plasmid pBat-2 (3) (containing the cDNA for batroxobin), amounts of batroxobin for studies. We intended to synthe pLS-D1 (7) (containing the synthetic tryptophan pro size batroxobin in E. coli by the use of recombinant DNA moter, polypeptide LH gene, somatomedin C gene, and fd techniques, which would be suitable for large-scale produc phage central terminator), pLS-T2 (7) (containing the tion. It is known that the recombinant polypeptides pro polypeptide LH gene and somatomedin C gene), and pCLaHtrp3t (8) (containing the polypeptide Cd-LH gene 1 To whom all correspondence and reprint requests should be ad and ƒ¿-hANP gene) were described previously. dressed. Preparation of Synthetic Genes-All oligonucleotides Abbreviations: Bat., batroxobin; SMC, somatomedin C; DTT, di were synthesized by the phosphoamidate method using an thiothreitol; PBS, phosphate-buffered saline; BSA, bovine serum automated DNA synthesizer (Applied Biosystems, Model albumin; RIA, radioimmunoassay; HPLC, high-performance liquid chromatography; ƒ¿-hANP, alpha-human atrial natriuretic poly 381A). The synthetic genes for the N-terminus of batrox peptide. obin and factor XIII-like polypeptide were constructed by

632 J. Biochem. Expression of cDNA for Batroxobin 633 the ligation of the uhosnhorvlated oligonucleotides (7. 8). with four volumes of cold acetone (-20•Ž). After centrifu Construction of Expression Plasmids-The constructions gation (-10•Ž, 18,000xg, 20 min), the precipitate was of pBat-SS3 and pBat-SS49 were performed as shown in rinsed gently with ice-cold 80% acetone. The precipitate Fig. 1. The direct expression plasmid, designated as was dried and then dissolved in 100mM Tris-HCl buffer pBat-SS3, was constructed as follows. Plasmid pLS-D1 was (pH8.0) containing 8M urea and 25mM EDTA. This digested with BamHI and both cohesive ends were subse solution was diluted with a refolding buffer consisting of 50 quently filled in with DNA polymerase I (Klenow frag mM Tris-HC1, 20% glycerol, 0.15 to 1.5M NaCl, 0.1mM ment). The linear plasmid was then digested with EcoRI, EDTA, and 0.01% BSA (bovine serum albumin). The and the resultant fragment was ligated to the batroxobin solution of recombinant batroxobin was allowed to stand at gene (RsaI-HpaI fragment from pBat-2) through the room temperature for approximately 20h. The refolded synthetic oligonucleotide. The fusion expression plasmid, batroxobin was examined by radioimmunoassy and/or designated as pBat-SS49, was constructed by ligation of the biological assay (see below). trp promoter-polypeptide Cd gene (pCLaHtrp3t cleaved Radioimmunoassay (RIA) of BatroxobinA 105-fold- with PstI/EcoRI) to the pBat-SS3 cleaved with PstI/Rsal diluted solution (100pl) of anti-batroxobin serum (raised through the synthetic oligonucleotide encoding the factor in rabbit) prepared in the assay buffer (PBS containing XIII-like polypeptide. 0.5% BSA and 25mM EDTA) and 100pl of 125I-labeled Two-cistron expression plasmids, designated as pBat- batroxobin (approximately 105cpm/ml) prepared by the SS5 and pBat-SS8, were constructed as follows. Plasmid Iodogen method (12) were added to 500ƒÊl of the renatured pBat-SS5 was constructed by inserting the batroxobin gene batroxobin solution or to the batroxobin standard solution (consisting of the synthetic oligonucleotide for the direct in the assay buffer. After incubation at 4•Ž overnight, 100 expression plasmid and Rsal-HpaI fragment from pBat-2) pl of normal rabbit serum, 100pl of anti-gamma globulin between trp promoter-SD sequence and somatomedin C serum (prepared from rabbit) and 900ƒÊ1 of 5% polyethyl gene of the pLS-D1. Plasmid pBat-SS8 was constructed by ene glycol 6000 were added. The solution was left standing ligation of the batroxobin gggggene(PstI-EcoRI fragment from at 4•Ž for 2.5h, and then centrifuged at 4•Ž, 3,000xg for pBat-SS3) downstream to the trp promoter-somatomedin 30min. The radioactivity of the pellet was counted with an C gene (PstI-EcoRI partially digested fragment from auto-gamma-counter (Packard, model 800). pLS-T2). Assay of Enzymatic Activity of Batroxobin on Fibrinogen- All DNA manipulations were carried out essentially as Agarose Plates-Modified fibrinogen-agarose plates (1) described by Maniatis et al. (9). Constructed plasmids were prepared as follows: 50mg of agarose was dissolved in were characterized by restriction endonuclease digestion, 5ml of PBS at 90•Ž and the solution was cooled to 45•Ž. and the nucleotide sequence of the synthetic genes was Five milliliters of 0.4% bovine fibrinogen (plasminogen- verified by the Maxam-Gilbert method (10). free, Miles Scientific, Naperville) in PBS, prewarmed to Expression and Preparation of Recombinant Batroxobin 45•Ž, was added and the mixture was immediately poured -Bacterial transformants were grown in 400ml of M9 into a dish and allowed to gel. A 10ƒÊl aliquot of batroxobin liquid media containing casamino acids (0.5%, w/v) and solution was applied to fibrinogen-agarose plates and in ampicillin (25pg/ml) at 30•Ž to an absorbance of 0.6 at 600 cubated in a humid atmosphere overnight at 37•Ž in order nm. 3-Indoleacrylic acid (IAA) was then added to a final to cause clot propagation. As batroxobin is capable of concentration of 10pg/ml to derepress the trp promoter. converting fibrinogen to fibrin due to the thrombin-like Incubation was continued for an additional 2.5h, and the activity, the formation of fibrin was observed as distinct cells were harvested by centrifugation at 7,000xg for 10 turbidity. The diameter of each clot was measured by min at 4•Ž. Pellets were suspended in 8ml of phosphate means of a micrometer device and recorded on a semi buffered saline (PBS, pH 8.0) containing 25mM EDTA, logarithmic scale as a function of the batroxobin concentra and sonicated for 2 min in an ice bath. After centrifugation tion in the solution. at 18,000xg for 20 min at 4•Ž, the insoluble pellets Other Techniques-In vitro translation was carried out (PBS-insoluble fraction) were resuspended in 10ml of 50% using a Prokaryotic DNA-directed translation kit (Amer (v/v) glycerol and sonicated, then centrifuged as described sham, Tokyo) according to the procedure recommended by above. The pellets, after being washed with 50% (v/v) the manufacturer. 35S-Labeled proteins were visualized by glycerol twice more, were suspended in 4ml of 100mM SDS-PAGE followed by fluorography. HPLC was per Tris-HCl buffer (pH8.0) containing 8M urea and 25mM formed under the following conditions: column, YMC EDTA and sonicated for 2 min in an ice bath. After AP-302 (4.6x75mm); detection, absorbance measure centrifugation at 20,000xg for 20 min at 4•Ž, the super- ment at 210nm; flow rate, 1.0ml/min; elution solvent, natants were collected. 0.1% trifluoroacetic acid with a 10-60% acetonitrile gradi Cleavage of the Fusion Protein with Thrombin-The ent. The amino acid sequence was determined using a gas solution of partially purified fusion proteins (5ƒÊl) was phase sequencer (Applied Biosystems, model 470A). The treated with thrombin (5 to 8 units) in 40ƒÊl of 10mM protein concentration was estimated from the absorbance Tris-HC1 buffer (pH 8.0) containing 1M urea, at 37•Ž for 6 at 280nm assuming that one absorbance unit corresponds h. to 0.5mg protein/ml. Purification and Refolding to Recombinant Batroxobin -Samples digested with thrombin were subjected to RESULTS AND DISCUSSION preparative 15% SDS-PAGE (11). Recombinant batrox. obin was separated from the gel by electroelution using Construction of the Expression Plasmids and Production Max Yield (Atto, Tokyo) according to the procedure recom of Batroxobin-In order to effect the expression of mature mended by the manufacturer, and then was precipitated batroxobin, we first constructed the direct expression

Vol. 109, No. 4, 199Vol.109,No.4, 1991 634 M. Maeda et al. plasmid (pBat-SS3, Fig. 1). However, the production of Both pBat-SS5 and pBat-SS8 contain the batroxobin gene batroxobin was not detectable by SDS-PAGE or radioim and a fused gene made up of somatomedin C gene and a part munoassay (data not shown). Further, the translation of (corresponding to Cys1-Leu59, designated as polypeptide batroxobin mRNA was not detected in an in vitro transla LH) of the interferon-gamma gene in two-cistron systems tion system (data not shown). downstream from the trp promoter. They are different with The construction of the plasmids for the two-cistron respect to the position of the batroxobin gene. Neither system and fusion protein method is summarized in Fig. 2. pBat-SS5 nor pBat-SS8 produced the mature batroxobin in

Fig. 1. Schematic outline of the construction of the expression plasmids. Batroxobin coding sequences are represented by the open box. The shaded box represents the synthetic trp promoter region. The double-hatched box represents the factor XIII-like sequence. The hatched box represents the synthetic fd phage central terminator. Amp' indicates the ƒÀ-lactamase gene. The solid thick line represents the polypeptide Cd gene, the polypeptide LH, the somatomedin C gene, or the ƒ¿-hANP gene. The thin single line represents the pBR322 sequences. In order to effect the expression of mature batroxobin, pBat-SS3 was constructed as follows; the 5•L-noncoding region and a part of the coding region for the signal peptide and zymogen peptide were replaced by the synthetic gene coding for the N-terminal region of muture batroxobin.

Fig. 2. Summary of the structure of constructed plasmids and their capac ity for expressing a batroxobin gene. The column headed expression indicates the results from in vitro translation experi ments. Translation of protein from each plasmid is given as detectable (+) or not detectable (-).

J. Biochem. Expression of DNA for Batroxobin 635

the in vitro translation system (data not shown). However, sion plasmid, designated as pBat-SS49, was used to trans these plasmids (pBat-SS5 and pBat-SS8) produced fusion form E. coli HB101-16. The bacterial transformants were proteins, LH-SMC, efficiently with nearly the same yield. cultivated in M9 liquid media containing casamino acids to The results suggest that the failure of expression of the produce the fused batroxobin (Fig. 3, approximately 40mg batroxobin gene is a post-transcriptional event probably of the fused batroxobin was produced in 1 liter of culture due to the formation of secondary structure of the mRNA broth). The produced fusion polypeptide was aggregated to unsuitable for the translation of batroxobin in E. coli. form inclusion bodies (5) which were clearly seen by the Finally we constructed the plasmid which is expected to use of a phase-contrast microscope (data not shown). encode a fusion protein composed of batroxobin and a part We constructed other expression plasmids to produce (Phe23-G1u46, designated as polypeptide Cd) of somato fusion proteins using the synthetic genes encoding poly medin C. This polypeptide has been used effectively as a peptide LH or polypeptide Cd-LH (described previously; partner to form fusion proteins with somatomedin C (7, 13) 7, 8). When fused with these small peptides batroxobin was or ƒ¿-hANP (8). Moreover, to liberate mature batroxobin produced in sufficient amounts. The reason why the batrox from the fusion protein, we introduced an oligonucleotide obin could only be expressed as a fusion protein, is unclear. corresponding to a peptide with the thrombin recognition It is suggested that the fused polypeptide genes (poly sequence between the Cd and batroxobin genes. Thus the peptides LH, Cd, and Cd-LH) change the environment of produced fusion protein possesses the specific thrombin the 5•L end of the mRNA and this causes disruption of the cleavage site between the C-terminal of polypeptide Cd and stable secondary structure. Further the fused polypeptides the N-terminal of the mature batroxobin. may be able to stabilize batroxobin in E. coli. We constructed the expression plasmid for the produc Isolation and Purification of Batroxobin Generated from tion of mature batroxobin based on the above considera tions (Fig. 1). The peptide contains the cleavage site for thrombin. The synthetic oligonucleotide encoding the tetra decapeptide Met-Asp-Asp-Leu-Pro-Thr-Val-Glu-Leu-Gln- Val-Val-Pro-Arg was inserted between the polypeptide Cd gene and the mature batroxobin gene. The resulting expres-

Fig. 3. SDS-PAGE analysis of E. coli lysates. E. coli HB101.16 containing pBat-SS49 (lanes 1 and 2) was grown under inducing con ditions, and then was lysed by sonication. Lane 1, cellular sol uble proteins; lane M, molecular weight markers (top to bottom, 92,500, 66,200, 45,000, 31,000, 21,500, 14,400); lane 2, cellular insoluble proteins. The arrow in dicates the fused batroxobin con taining factor XIII-like sequence Fig. 5. SDS-PAGE analysis (A) and HPLC profile (B) of the present in the PBS-insoluble frac recombinant mature batroxobin purified by preparative elec- tion. For SDS-PAGE, 15% gel was trophoresis. For SDS-PAGE, 15% gel was used. HPLC conditions used. are described in "MATERIALS AND METHODS."

Fig. 4. Cleavage of the fusion protein produced from E. coli harboring pBat-SS49 with thrombin. The solubilized fusion protein (P-SS49) was cleaved with different concentrations of thrombin in various concentra tions of urea. Cellular insoluble proteins were washed three times with 50% (v/v) glycerol, dissolved in 8 M urea, 100mM Tris-HCl, pH 8.0, 25mM EDTA, and then centrifuged at 20,000xg for 20 min. The supernatants (5ƒÊ1) were treated with thrombin in 40ƒÊ1 of 10mM Tris-HCl buffer (pH 8.0), at 37•Ž for 6h. Lane M shows molecular weight markers (top to bottom, 92,500, 66,200, 45,000, 31,000, 21,500, 14,400).

Vol. 109, No. 4, 1991 636 M . Maeda et al.

Fig. 7. Detection of the biological activity of refolded recom- binant mature batroxobin on a fibrinogen-agarose plate. A 10ƒÊ1 sample of the refolded batroxobin in the modified dilution buffer was applied to a fibrinogen-agarose plate and incubated overnight at 37•Ž.

mature batroxobin (3). These results show that the cleavage with thrombin occurred at the expected specific site. All the recombinant proteins containing the factor XIII-like sequence were correctly cleaved with thrombin irrespective of the type of the polypeptides fused (polypeptides LH, Cd, or Cd-LH) Fig. 6. Refolding of the recombinant batroxobin. (A) A solution (data not shown). It seems that thrombin recognized and of the purified mature batroxobin (500kg/ml) was diluted to several cleaved the factor XIII-like sequence of the fusion protein concentrations (2.5-25pg/ml) with dilution buffer consisting of 50 faster than it might cleave the putative recognition se mM Tris-HCl, pH 8.0, 150mM NaCl, 20% glycerol, 0.1mM EDTA, quences for thrombin located in the batroxobin sequence. and 0.01% BSA, and then the solution was left standing at room Most likely, the insertion of 14 amino acid residues contain temperature for about 20 h. Refolding was followed by assaying ing the factor XIII-like sequence may have contributed to batroxobin with RIA. (B) The solution containing 2.5kg of batroxobin our success. This approach may be applicable to other in 1 ml was adjusted to pH 6-10.5 with 1M HCl or 1M NaOH and refolded at room temperature for about 20h. (C) The same concentra large-molecular recombinant fusion proteins containing tion of batroxobin was adjusted to various NaCl concentrations in the many lysine and residues. range of 0-1.5M. Refolding of the Recombinant Batroxobin Produced in E. coli-We tried to refold the recombinant protein so as to obtain bioactive batroxobin. Although there are no estab the Fusion Protein-The fused batroxobin was refolded in lished refolding methods for recombinant proteins pro the redox buffer which contained reduced glutathione and duced in E. coli up to the present, several methods have oxidized glutathione (14). However, the refolded protein been used to refold many recombinant proteins. Solubiliza exhibited no reactivity towards the specific (data tion of recombinant proteins in buffer free of denaturing not shown). Therefore, the refolding was applied to the agents was achieved by dialysis or by dilution. The mature batroxobin obtained from the fused polypeptide. efficiency of this renaturation process may depend on such Thus, the fusion protein synthesized from pBat-SS49, factors as the concentration and purity of the recombinant designated as P-SS49, was treated with thrombin in 10 mM polypeptide, pH, ionic strength of the medium and presence Tris-HC1 buffer (pH 8.0) containing 8, 4, 2, and 1 M urea, or absence of thiol reagents (Refs. 15 and 16 for reviews). respectively, at 37•Ž for 6h (Fig. 4). P-SS49 could be We examined several methods (data not shown). We completely digested with 10 units of thrombin per 40ƒÊl of selected Hagers' method (17) for the refolding of the the reaction mixture containing 1M urea. On purification of recombinant batroxobin, as the refolding efficiency was mature batroxobin by preparative electrophoresis (the comparatively high. recovery of mature batroxobin from the polyacrylamide gel Refolding of the recombinant batroxobin in the dilution was about 70%), the cleavage product was obtained as a buffer without a thiol reagent such as dithiothreitol (DTT) homogeneous preparation, as shown by SDS-PAGE analy was firstly tried. The refolding efficiency was increased sis (Fig. 5). The amino acid sequence from the N-terminal about 20-fold compared to that in the presence of DTT. of the purified protein was determined. The sequence, Since the recombinant mature batroxobin had been purified Val-Ile-Gly-Gly-Asp, corresponded to that of the native by preparative SDS-PAGE under reducing conditions, the

J. Biochem. Expression of cDNA for Batroxobin 637

thiol reagent might not be necessary. In the following experiment, this dilution buffer without DTT was used. The REFERENCES refolding efficiency of batroxobin was highly dependent on 1. Stocker, K. (1976) in Methods in Enzymology (Lorand, L., ed.) the concentration of the recombinant batroxobin, pH and Vol. 45, pp. 214-223, Academic Press, New York ionic strength of the medium: the optimum conditions for 2. Stocker, K. (1978) in Handbook of Experimental Pharmacology the refolding of recombinant batroxobin were a concentra (Markwardt, F., ed.) Vol. 46, pp. 451-484, Springer-Verlag, tion of 1-5ƒÊg/ml, pH 6.5-8.5, and 0.5-1.3M NaCl, pH Berlin 7.5, as shown in Fig. 6. 3. Rob, N., Tanaka, N., Mihashi, S., & Yamashina, I. (1987) J. Biol. The biological activity of the recombinant batroxobin Chem. 262, 3132-3135 was assayed by measuring its ability to convert fibrinogen 4. Itoh, N., Tanaka, N., Funakoshi, I., Kawasaki, T., Mihashi, S., & Yamashina, I. (1988) J. Biol. Chem. 263, 7628-7631 to fibrin. It showed thrombin-like activity on a fibrinogen 5. Kane, J.F. & Hartley, D.L. (1988) Trends Biotechnol. 6, 95-101 agarose plate (Fig. 7). The specific activity (biological 6. Saito, Y., Sasaki, H., Hayashi, M., Notani, J., Kobayashi, M., & activity/immunoreactivity assayed by RIA) of the batrox Niwa, M. (1989) Eur. Pat. Appl. 302456 obin derived from the fusion proteins was 60-84% of that of 7. Sato, S., Kusunoki, C., Saito, Y., Niwa, M., & Ueda, I. (1987) mature batroxobin. The lower specific activity may be Eur. Pat. Appl. 219814 because partially refolded batroxobin with no or little 8. Saito, Y., Ishii, Y., Koyama, S., Tsuji, K., Yamada, H., Terai, T., biological activity is recognized by polyclonal . Kobayashi, M., Ono, T., Niwa, M., Ueda, I., & Kikuchi, H. (1987) J. Biochem. 102, 111-122 The low recovery (about 3%) of batroxobin activity as a 9. Maniatis, T., Fritsch, E.F., & Sambrook, J. (1982) Molecular result of the low efficiency of the refolding is a problem Cloning, Cold Spring Harbor Laboratory, New York which remains to be solved. 10. Maxam, A.M. & Gilbert, W. (1980) in Methods in Enzymology These findings provide a basis for producing a large (Grossman, L. & Moldave, K., eds.) Vol. 65, pp. 499-560, amount of the recombinant protein. The availability of Academic Press, New York 11. Laemmli, U.K. (1970) Nature 227, 680-685 large quantities of bioactive batroxobin produced in E. coli 12. Salacinski, P.R., McLean, C., Sykes, J., Clement-Jones, V.V., & will make possible a number of interesting biochemical Lowry, P.J. (1981) Anal. Biochem. 117, 136-146 experiments. 13. Saito, Y., Yamada, H., Niwa, M., & Ueda, I. (1987) J. Biochem. 101,123-134 We thank Drs. Y. Saito and M. Kobayashi of our group for helpful 14. Harris, T.J.R., Patel, T., Marston, F.A.O., Little, S., Emtage, discussions and T. Tamura in our laboratories for amino acid sequence J.S., Opdenakker, G., Volckaert, G., Rombauts, W., Billiau, A., analysis. & De Somer, P. (1986) Mol. Biol. Med. 3, 279-292 15. Light, A. (1985) BioTechniques 3, 298-306 16. Marston, F.A.0. (1986) Biochem. J. 240,1-12 17. Hager, D.A. & Burgess, R.R. (1980) Anal. Biochem. 109, 76-86

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