Mol Biotechnol DOI 10.1007/s12033-015-9845-6

RESEARCH

New Strategies for Expression and Purification of Recombinant Human RNASET2 Protein in Pichia pastoris Marta Lualdi • Edoardo Pedrini • Francesca Petroni • Johnny Na¨sman • Christer Lindqvist • Debora Scaldaferri • Roberto Taramelli • Antonio Inforzato • Francesco Acquati

Ó Springer Science+Business Media New York 2015

Abstract Ribonucleases form a large family of enzymes involved in RNA metabolism and are endowed with a broad range of biological functions. Among the different RNase proteins described in the last decades, those belonging to the Rh/T2/S subfamily show the highest degree of evolutionary conservation, suggesting the occurrence of a key critical ancestral role for this protein family. We have recently defined the human RNASET2 gene as a novel member of a group of oncosuppressors called ‘‘tumor antagonizing genes,’’ whose activity in the control of cancer growth is carried out mainly in vivo. However, to better define the molecular pathways underlying the oncosuppressive properties of this protein, further structural and functional investigations are necessary, and availability of high-quality recombinant RNASET2 is of paramount importance. Here, we describe a multi-step Marta Lualdi and Edoardo Pedrini have contributed equally to the manuscript.

Electronic supplementary material The online version of this article (doi:10.1007/s12033-015-9845-6) contains supplementary material, which is available to authorized users. M. Lualdi  E. Pedrini  D. Scaldaferri  R. Taramelli  F. Acquati (&) Department of Theoretical and Applied Sciences, University of Insubria, Via JH Dunant 3, 21100 Varese, Italy e-mail: [email protected] M. Lualdi e-mail: [email protected] E. Pedrini e-mail: [email protected] D. Scaldaferri e-mail: [email protected] R. Taramelli e-mail: [email protected]

strategy that allows production of highly pure, catalytically competent recombinant RNASET2 in both wild-type and mutant forms. The recombinant proteins that were produced with our purification strategy will be instrumental to perform a wide range of functional assays aimed at dissecting the molecular mechanisms of RNASET2-mediated tumor suppression. Keywords RNase expression  IMAC purification  SEC analysis  IEX chromatography  Protein tagging  Deletion mapping  Ribonuclease activity  Deglycosylation assay

Introduction Ribonucleases (RNases) are evolutionary conserved enzymes endowed with a pivotal role in determining cell life or death. Indeed, the key role that RNA turnover plays in cellular metabolism is demonstrated by both the impressive number of enzymes involved in RNA hydrolysis deployed by prokaryotic and eukaryotic cells and the wide range of biological processes controlled by F. Petroni  A. Inforzato (&) Laboratory of Immunopharmacology, Humanitas Clinical and Research Center, Via Manzoni 56, 20089 Rozzano, MI, Italy e-mail: [email protected] F. Petroni e-mail: [email protected] J. Na¨sman  C. Lindqvist ˚ bo Akademi University, Department of Biosciences, A Tykistokatu 6, 20520 Turku, Finland e-mail: [email protected] C. Lindqvist e-mail: [email protected]

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these enzymes [1]. Ribonucleases catalyze the cleavage of a variety of RNA substrates, such as single-stranded or double-stranded RNA and RNA–DNA hybrid duplexes. Both exo- and endo-ribonucleases have been described, and the latter have recently stimulated a particular interest due to their widespread functions [2]. Moreover, several RNases are known to be actively secreted by cells or targeted to selected intracellular compartments, where they are involved in specific biological roles. These enzymes mostly include members of the A, T1, and T2 families of ribonucleases, and a particular interest has been recently focused on RNases belonging to the latter class (Rh/T2/S ribonucleases), which represents a wide group of acid endo-ribonucleases that cleave single-stranded RNA and are either located in specific subcellular compartments or directly secreted by cells [3]. Enzymes of the T2 family have a molecular mass around 25 kDa, display no significant base specificity, and carry two highly conserved active-site segments (CAS I and CAS II) that are required for their catalytic activity [3]. Strikingly, unlike T1 and A RNases, a member of the T2 RNases family can be found in almost every organism including plants, fungi, bacteria, viruses, and animals, suggesting an important and ancestral function for these enzymes [3]. The RNASET2 gene represents the unique human member of the T2 ribonuclease family [4]. The encoded 256 aminoacid preprotein includes a N-terminal signal peptide for secretion, a catalytic core, and a C-terminal portion. Very recently, the 3D structure of the human RNASET2 protein has been determined by X-ray crystallography [5]. The structure shows typical features for members of the T2 RNase family, with an a ? b motif made by a seven a-helices plus eight b-strands module. By contrast, the C-terminal region between Cys213 and Val240 clearly distinguishes human RNASET2 from its non-human orthologs, suggesting a potential role for this region in our species [5]. Interestingly, in silico analysis of this region using a tool for the identification of functional linear motifs predicted a potential TRAF2-binding motif (PKQE, residues 222–225 of the preprotein sequence), which might confer a crucial functional role to RNASET2 in the regulation of cell survival and apoptosis [5]. Recent experimental evidence by our research group reported that the RNASET2 gene acts as a tumor antagonizing gene in an ovarian cancer model and that this role is not dependent on the catalytic activity of the protein [6, 7]. Moreover, our data strongly supported the hypothesis that RNASET2-mediated tumor suppression is carried out in the context of the cancer microenvironment. Indeed, the recruitment and activation of cells belonging to the monocyte-macrophage lineage were shown to be crucial for the observed in vivo oncosuppressive role of RNASET2 [7, 8].

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In order to further investigate the mechanisms of RNASET2-mediated tumor suppression, the role of this protein in establishing a functional cross-talk between cancer cells and the tumor microenvironment must be thoroughly investigated. Moreover, since the catalytic activity is dispensable for the tumor suppressive function of RNASET2, functional mapping of the protein is required to identify domains and/or motifs involved in its oncosuppressive role. To shed light on these issues, the availability of highly pure recombinant RNASET2 protein is an essential requirement. To this purpose, we have recently set up protocols for expression and purification of recombinant RNASET2 protein in two different heterologous expression systems: the yeast Pichia pastoris and the baculovirus/insect cells system (BEVS) [9]. In both systems, a catalytically competent RNASET2 protein was produced, with comparable levels of expression (about 30 mg of protein/l of conditioned medium). The P. pastoris system proved to be more advantageous, being more cost-effective with respect to BEVS and allowing an easier purification of the protein by affinity chromatography from a protein-free supernatant [9]. However, a full exploitation of this research tool was precluded by several limitations inherent to both systems, such as: (1) protein heterogeneity (mostly in the BEVSderived material) due to high molecular weight species (mostly host cell proteins) co-eluting with the RNASET2 molecule; (2) a significant loss of the recombinant protein in both flow-through and wash fractions during affinity purification steps; (3) the presence of both mildly (38–45 kDa) and highly (50–80 kDa) glycosylated forms of the protein in the eluted fractions (mainly in RNASET2 preparations from P. pastoris); and (4) the occurrence of endotoxin contamination, which precluded the use of the recombinant protein in functional assays to be carried out with cells from the monocyte/macrophage lineage, which likely represent the most relevant target of the RNASET2 protein in vivo [7, 8]. In the present work, we report a significant implementation of the purification strategies for recombinant RNASET2 in order to overcome the above listed limitations. Moreover, we present data on the cloning, expression, and purification of a specific mutant form of recombinant human RNASET2 which will be instrumental to carry out a detailed functional map for this protein.

Materials and Methods Reagents and Media The EasySelectTM Pichia Expression Kit was available from Invitrogen (Thermo Fischer Scientific, Waltham, MA,

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USA). PhusionÒ High-Fidelity DNA Polymerase and restriction/modification enzymes were from New England Biolabs (NEB, Ipswich, MA, USA). Zeocin was purchased from InvivoGen (InvivoGen, Toulose, France) and Yeast Nitrogen Base from Difco BD (Becton–Dickinson and Company, Franklin Lakes, NJ, USA). Bradford reagent and Polyprep columns were from BioRad (Bio-Rad laboratories, Hercules, CA, USA). Ultrafree centrifugal concentrators (5 kDa MWCO) were from Merck Millipore (EMD Millipore, Billerica, MA, USA), VivaFlow 50 cross-flow cassettes (10 kDa MWCO) from Sartorius (Sartorius AG, Goettingen, Germany), and Slyde-A-Lyzer dialysis cassettes (10 kDa MWCO) from Thermo Scientific (Thermo Fischer Scientific, Waltham, MA, USA). Ni–NTA resins were obtained from Qiagen (Qiagen N.V., Venlo, Netherlands); HisTrap HP (1 ml), HiTrap Q HP (1 ml), and Superdex 75 10/300 GL columns were from GE Healthcare (General Electric Co., Fairfield, CT, USA). NuPAGE Bis–Tris precast gels were from Life Technologies (Thermo Fischer Scientific, Waltham, MA, USA); nitrocellulose and PVDF membranes were from GE Healthcare (General Electric Co., Fairfield, CT, USA). A rabbit polyclonal antibody was raised against the fulllength wild-type human RNASET2 recombinantly made in a baculovirus–insect cells system [4] and affinity purified by Davids Biotechnologie (Davids Biotechnologie GmbH, Regensburg, Germany). The mouse anti-HA tag monoclonal antibody (clone I2CA5) was purchased from Roche (F. Hoffmann-La Roche SA, Basel, Switzerland), and the mouse anti-His tag monoclonal antibody was from Life Technologies (Thermo Fischer Scientific, Waltham, MA, USA). Horseradish peroxidase-conjugated secondary antibodies and SuperSignalÒ West Dura Extended Duration Chemiluminescent Substrate were available from Thermo Scientific (Thermo Fischer Scientific, Waltham, MA, USA). All other reagents were from Sigma-Aldrich (SigmaAldrich Corporation, St. Louis, MO, USA), unless otherwise stated. Cloning of the Human RNASET2 Coding Sequence in Plasmid Vectors Cloning of both wild-type (wtRNASET2) and catalytically inactive (RNASET2 H65F/H118F) RNASET2 coding sequences, fused at the 30 -end to sequences coding for both HA (hemagglutinin) and 69 Histidine tags, into the pPICZaA expression vector has been described previously (pPICZaA-wtRNASET2 and pPICZaA-RNASET2 H65F/ H118F) [9]. A mutant version of RNASET2 (delTRAF2bd_RNASET2His) deleted of 4 aa (PKQE, residues 222–225 of the preprotein

sequence) representing a predicted TRAF2 binding domain was cloned in pPICZaA (pPICZaA-delTRAF2bdRNASET2). Overlap-extension PCR was performed using pPICZaAwtRNASET2 as a template. Sequences (50 –30 ) of the applied primer pairs are: RNASET2 fw Eco: TAAGAATTCTCTCCGCAGGTC GGCACC RNASET2 delTRAF2 rev: AGCCAGACGGACGGCT GCTCCCCCGGCT RNASET2 delTRAF2 fw: AGCCGTCCGTCTGGCTG GCAAATGGGGC RNASET2 XhoI rev: TGACCTCGAGTCAATGCTTG GTCTTTTTAGGTGGT The amplification product was digested with EcoRI and XhoI, gel purified, and cloned into pPICZaA before transforming in the DH5a E. coli strain. Plasmid DNA was purified, and, after sequencing (BMR, Padova, Italy), the empty vector and pPICZaA-delTRAF2bdRNASET2 construct were used to transform X33 P. pastoris strain. The same protocol was used for cloning of the wild-type RNASET2 coding sequence with an additional 69 His tag at the 50 -terminus (pPICZaA-wt2tag RNASET2). The pPICZaA-wtRNASET2 construct was used as a template for a PCR reaction with the following primer pair (50 –30 ): RNASET2 fw Eco69 His 25aa: ACTGGAATTCCATCACCACCATCATCACGACAA GCGCCTGCGTGAC 30 AOX rev: GCAAATGGCATTCTGACATCC Transformation of P. pastoris and ‘‘methanol utilization’’ Phenotype Screening RNASET2-coding constructs or empty vectors were linearized within the 30 AOX region with PmeI restriction enzyme and transformed into P. pastoris by the lithium chloride method [10]. The methanol utilization plus (Mut?) X33 strain was transformed. Transformants were selected on yeast extract peptone dextrose plates (1 % yeast extract, 2 % bactopeptone, 2 % dextrose, 20 g/l bactoagar) containing 100 lg/ml zeocin and confirmed by streaking on the same plates. Zeocin-resistant clones were picked and lysed by boiling at 100 °C for 10 min. PCR reactions were performed on lysates according to manufacturer’s instructions in order to discriminate integration of the empty vector from integration of RNASET2containing constructs. Mut? strain colonies positive for integration were also screened for the methanol utilization phenotype by streaking on minimal dextrose histidine and minimal methanol histidine plates (MDH and MMH: 1.34 % yeast nitrogen base, 4 9 10-5 % biotin, 0.004 % histidine, 15 g/l bactoagar, and either 2 % dextrose for MDH or 0.5 % methanol for MMH plates).

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Expression of RNASET2 in P. pastoris Buffered complex glycerol or buffered complex methanol (BMGY/BMMY) (1 % yeast extract, 2 % bactopeptone, 1.34 % yeast nitrogen base, 4 9 10-5 % biotin, and either 1 % glycerol or 0.5 % methanol) media were used to grow cells and analyze the expression of RNASET2. Briefly, selected clones were plated and grown for 3 days at 28 °C. One colony was picked and inoculated into 50 ml of BMGY medium. Cultures were grown at 28 °C overnight until they reached an optical density at 600 nm (OD600) between 2 and 6. They were then centrifuged, and the cell pellet was resuspended in BMMY at a starting OD600 of 1 for induction of protein expression. Fresh methanol (0.5 % final concentration) was added to the culture each day, from time 0 to 7 days from the first induction. Expression of RNASET2 in BEVS Expression and purification of recombinant RNASET2 protein from BEVS supernatants were performed following previously described protocols [4, 9]. Protein Purification Cell cultures were processed after 7 days from induction, and the best producer clone was selected to express the protein for subsequent purification. After pelleting the cells at 2,500 g in a swinging bucket rotor, the supernatant was filtered (0.45 lm) and processed. Gravity-flow affinity chromatography was performed as described previously [4, 9]. Briefly, conditioned media were concentrated (10 kDa cut-off) and then incubated with the Ni–NTA Agarose matrix, in the presence of 10 mM imidazole (Qiagen). The post-binding mixtures (medium/resin) were poured into columns. The flowthrough, wash (50 mM imidazole) and eluate fractions (250 mM imidazole) were collected and analyzed by SDSPAGE (silver staining) and immunoblotting. For FPLC purification, conditioned media (500 ml to 1 l volumes) from both P. pastoris and BEVS cultures expressing RNASET2 proteins were concentrated by ultrafiltration on VivaFlow 50 cross-flow cassettes (10 kDa MWCO) to final volumes of 20–50 ml. The concentrated solutions were extensively dialyzed against PBS (20 mM NaPhosphate, 150 mM NaCl, pH 7.50) using Slyde-ALyzer dialysis cassettes (10 kDa MWCO) and adjusted to 500 mM NaCl and 10 mM Imidazole final concentrations prior to loading onto a HisTrap HP (1 ml) column equilibrated with PBS containing 500 mM NaCl and 10 mM Imidazole (equilibration buffer). Retained proteins were eluted at 1 ml/min using a step gradient of Imidazole in equilibration buffer.

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Eluates from the HisTrap column were further purified by negative-mode anion exchange chromatography (i.e., RNASET2 proteins passed through the stationary phase and contaminants were retained on it). To this end, protein solutions from HisTrap were made 100 mM NaCl by dilution with 20 mM Tris-Cl, pH 7.50, then loaded onto a HiTrap Q XL (1 ml) column equilibrated with buffer A (20 mM Tris-Cl, 100 mM NaCl, pH 7.50) at 1 ml/min, and eluted with a step gradient of buffer B (20 mM Tris– Cl, 1 M NaCl, pH 7.50). Where described, aliquots of both manually (i.e., from Ni–NTA resin) and FPLC (i.e., from either HisTrap or HiTrap Q columns) purified proteins were chromatographed on a Superdex 75 10/300 GL gel filtration column, using PBS as eluent at 0.5 ml/min. In all cases, chromatography columns were mounted onto and ¨ KTA Purifier FPLC system, and protein operated by an A elution was monitored as UV absorbance at 280 nm. Once homogenous preparations were obtained for each RNASET2 form (as assessed by analytical SEC, SDS-PAGE, and immunoblotting analyses), protein concentration was determined by UV absorbance at 280 nm using an average value of 66,000 M-1 cm-1 for the extinction coefficient of the different recombinant proteins (i.e., as computed by the ProtParam algorithm [11]). Standard curves of the purified proteins were assembled in immunoblotting experiments, and concentration of RNASET2 protein in both conditioned media and chromatography fractions was assessed by densitometry using the ImageJ software. Endotoxin Detection Recombinant RNASET2 proteins, purified as described above, were assessed for endotoxin contamination with the PYROGENTTM Gel Clot LAL Single Test Vials kit (Lonza) which has a sensitivity of 0.125 EU/ml, according to the manufacturer’s instructions. In all cases, protein concentration was adjusted to 100 lg/ml by dilution with endotoxin-free water, and an endotoxin titer of 0.125 EU/ ml/100 lg proteins was set as a threshold, based on the range of RNASET2 concentrations (10–100 lg/ml) required for functional assays on monocytes/macrophages. Analysis of RNASET2 Expression by Immunoblotting Aliquots of conditioned media from RNASET2-expressing P. pastoris and BEVS cultures as well as a relevant fraction from chromatography were separated on either 4–12 % and 10 % Bis–Tris or 13 % Tris–Glycine gels. Proteins were either revealed by Silver Staining (Sigma-Aldrich) or transferred onto PVDF/nitrocellulose membranes. These were saturated with either PBS or Tris-buffered saline containing 5 % (w/v) non-fat dry milk, and RNASET2 proteins were revealed with selected primary antibodies

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(i.e., either rabbit anti-RNASET2 polyclonal antibody, mouse anti-HA tag monoclonal antibody or mouse anti-His tag monoclonal antibody) followed by the appropriate horseradish peroxidase-conjugated anti-mouse or -rabbit IgG secondary antibody. Membranes were incubated with SuperSignalÒ West Dura Extended Duration Chemiluminescent Substrate, and chemiluminescence recorded on a ChemidocÒ MP system (Bio-Rad) or X-ray films (Thermo Scientific). Zymographic Analysis To assess the catalytic activity of recombinant RNASET2 proteins purified from both BEVS and P. pastoris supernatants, a zymographic analysis was performed as previously described [9]. Briefly, polyacrylamide gels were prepared with the addition of total RNA from Torula yeast in resolving (2 mg/ml) and stacking (0.3 mg/ml) gel solutions. Proteins were diluted in non-reducing sample buffer (125 mM Tris–HCl pH 6.8, 2 % SDS, 10 % glycerol, 0.02 % bromophenol blue) and loaded without boiling. After electrophoresis, SDS was removed with 25 % isopropanol in wash solution (10 mM Tris–HCl, pH 7.4); then gels were washed extensively and incubated at 51 °C in freshly prepared activity buffer (100 mM KCl, 100 mM Sodium acetate, pH 5.0). Enzymatic activity was detected by bleaching of the toluidine blue staining. After image acquisition, gels were completely destained and processed for immunoblotting. Deglycosylation The different forms of purified recombinant RNASET2 protein (200 ng/sample) were denatured at 95 °C for 5 min and either treated with peptide-N-glycosidase F (PNGase F, NEB) or mock treated, following the manufacturer’s instructions. Samples were then processed for immunoblotting as described above (40 ng protein/lane).

Results Gravity-Flow Affinity Coupled to Size-Exclusion Chromatography Protocols for the expression of recombinant human RNASET2 protein have been previously set up in our laboratory in two different heterologous expression systems, namely BEVS and the yeast P. pastoris [9]. For both systems, the recombinant RNASET2 protein was purified from cell culture supernatants by affinity gravity-flow chromatography (IMAC) using a matrix for purification of His-tagged

proteins, following previously described protocols [4, 9]. When the obtained fractions were assessed by SDS-PAGE and immunoblotting analysis, we found that the recovery rate of the recombinant protein was rather low due to protein loss in both flow-through and wash fractions (Table 1). Moreover, high molecular weight host-derived contaminants co-eluted with the recombinant protein, mostly in the RNASET2 preparations from BEVS (Fig. 1, lower panel). In addition, highly glycosylated forms of the recombinant protein, which clearly differed from the RNASET2 protein made in mammalian cells, were present in the P. pastoris supernatants [9] (Fig. 1, upper panel). Finally, significant endotoxin contamination levels (i.e., more than 0.125 EU/ml/100 lg proteins) were observed in both preparations by the LAL assay, which precluded the purified RNASET2 protein to be used in functional assays on monocytes/macrophages. As a first attempt to overcome these limitations, we combined the affinity gravity-flow purification protocol (IMAC) with a size-exclusion chromatography (SEC) polishing step (IMAC/SEC). SEC-FPLC was carried out on recombinant RNASET2 proteins that were isolated from both BEVS and P. pastoris by IMAC. As shown in Fig. 1, IMAC alone left some high molecular weight contaminants. More in detail, two discrete contaminant peaks were observed in the SEC chromatograms, paralleled by non-RNASET2 bands in silver-stained gels, from both wild-type (Fig. 1, lower panel) and catalytically impaired H65F/H118F [4] (Supplementary Fig. 1) recombinant RNASET2 proteins expressed in the BEVS system. On the other hand, an asymmetric fronting peak was observed in the chromatogram recorded for the wild-type RNASET2 protein purified from P. pastoris (Fig. 1, upper panel), mostly due to the presence of highly glycosylated forms of the protein (50–75 kDa). By contrast, when the RNASET2containing fractions from IMAC/SEC were pooled and assessed for purity by analytical SEC and SDS-PAGE, highly pure RNASET2 proteins were obtained (Fig. 1, Supplementary Fig. 1, solid lines). SEC polishing proved effective on BEVS-derived RNASET2, with relative yields of at least 80 % (Table 1). However, recovery of the P. pastoris protein from Superdex 75 was rather low (e.g., less than 50 %, see Table 1), likely due to poor resolution of RNASET2 from its higher molecular weight glycoforms on the applied chromatography column. Moreover, when RNASET2-containing fractions from both BEVS and P. pastoris systems were assessed for Gram-negative bacterial endotoxin contamination following IMAC/SEC (using the LAL gel clot assay), the test was still positive (i.e., more than 0.125 EU/ml/100 lg proteins), suggesting that endotoxin contamination remains an intrinsic limitation of this protein purification protocol.

123

123

ND b

Concentration of the RNASET2 proteins in conditioned media and IMAC eluates was determined by densitometric analysis of the corresponding immunoblots Concentration of the RNASET2 proteins from SEC and IEX was determined by spectrophotometry

ND ND ND ND ND ND ND 52 90 95 10.5 IEX

9.9b

52

7.0

6.3b

58 58 55 55 19.0 IMAC

10.5a

10.5 SEC

5.0

12.0

7.0a

ND ND 48

26

ND

ND

ND ND 55 55 19.0

10.5a

b

ND

ND

Total yield (%) Relative yield (%) Eluted protein (mg) Loaded protein (mg) Relative yield (%) Loaded protein (mg)

Eluted protein (mg)

Total yield (%)

delTRAF2bd_RNASET2-His (12 mg/l) wt_RNASET2-His (39 mg/l)

IMAC

a

40

ND ND ND ND ND

ND

ND

ND

80

50 50

2.3b 2.9 49 90 7.8

7.0

b

54 14.5

7.8a

54

5.7

2.9a

Total yield (%) Relative yield (%) Eluted protein (mg) Loaded protein (mg) Total yield (%) Relative yield (%) Eluted protein (mg) Loaded protein (mg)

wt_RNASET2-His (29 mg/l)a

BEVS

a a

P. pastoris Chromatography

Table 1 Comparison of IMAC, SEC, and negative-mode IEX for purification of the RNASET2 proteins from P. pastoris and BEVS

H65F/H118F_RNASET2-His (10 mg/l)a

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Fig. 1 Comparison of IMAC and IMAC/SEC purification of wt_RNASET2-His from P. pastoris and BEVS. Conditioned media from P. pastoris and BEVS expressing wt_RNASET2-His were passed through Ni–NTA agarose gravity-flow columns equilibrated and eluted as described in ‘‘Materials and Methods’’ section. Affinityisolated proteins from both P. pastoris and BEVS (upper and lower panels, respectively) were further separated on a Superdex 75 ¨ KTA Purifier FPLC system. (10 9 300 mm) column using an A Chromatograms were recorded as UV absorbance at 280 nm (IMAC, dotted lines). wt_RNASET2-His containing fractions were pooled, concentrated, and chromatographed again on the same SEC column (IMAC/SEC, solid lines). IMAC- and IMAC/SEC-purified proteins were run on 10 % gels under denaturing and reducing conditions. Representative silver-stained gels are shown in both upper and lower panels (1 lg of total proteins/lane)

Affinity Coupled to Negative-Mode Anion Exchange Chromatography In order to improve both yield and homogeneity of the recombinant RNASET2 protein, we developed an alternative purification protocol, which coupled affinity chromatography by IMAC to an ion exchange chromatography (IEX) step. Since the P. pastoris expression system was more cost-effective than BEVS and allowed purification of the recombinant protein from a clear protein-free supernatant, we implemented this optimization strategy in the P. pastoris system only. The IMAC purification step on P. pastoris supernatant was performed on an FPLC chromatographer using HisTrap HP columns, and the eluted wild-type RNASET2 protein was further purified on HiTrap Q HP anion exchange columns. In particular, a negative-mode IEX was performed, where the RNASET2 protein was collected in the flow-through (unbound fraction), and both high molecular weight contaminants and hyperglycosylated forms of RNASET2 were eluted in high

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Fig. 2 Negative-mode IEX of wt_RNASET2-His from P. pastoris. a wt_RNASET2-His was isolated from P. pastoris conditioned medium with HisTrap HP (1 ml) columns and further purified on HiTrap Q XL (1 ml) columns by negative-mode ion exchange chromatography (IEX). A representative IEX chromatogram is shown where UV absorbance at 280 nm and mobile phase conductivity are reported as solid and dotted lines, respectively (upper panel). Unfractionated material (IMAC eluate) and HiTrap Q fractions

(unbound, 300 mM and 1 M NaCl) were analyzed on a Superdex 75 (10 9 300 mm) column. An overlay of the corresponding UV profiles at 280 nm is shown (lower panel). b The same fractions were run on 4–12 % gradient gels under denaturing and reducing conditions. Proteins were revealed by either silver staining (upper panel) or western blotting (lower panel), using an anti-His monoclonal antibody (1 lg and 200 ng of total proteins/lane, respectively)

salt conditions (Fig. 2a, upper panel). Analytical SEC on both IMAC eluates and HiTrap Q HP fractions confirmed that IEX greatly improved homogeneity of the recombinant RNASET2 protein (Fig. 2a, lower panel), whose presence in the IEX unbound fraction was confirmed by SDS-PAGE followed by immunoblotting analysis. Furthermore, very little, if any, RNASET2 was lost in the high salt fractions from IEX (Fig. 2b; Table 1). Therefore, the combined IMAC/IEX purification strategy allowed us to improve the overall yield of recombinant RNASET2 with respect to the IMAC/SEC approach (52 vs 26 %), although protein recovery still remained quite low, due to a significant loss of protein in the IMAC unbound fraction (Table 1). Moreover, the level of endotoxin contamination dropped below the threshold value of the LAL test (i.e., 0.125 EU/ml/100 lg proteins) following the IMAC/IEX approach. It is worth noting that the anti-His tag monoclonal antibody used to detect recombinant RNASET2 seems to have a poor affinity for the hyperglycosylated forms of the protein (i.e., in the 50–80 kDa range), similarly to the anti-HA tag monoclonal antibody

(data not shown). This is possibly due to protein oligosaccharides masking the recombinant tags to recognition by their respective antibodies. By contrast, the high molecular weight glycoforms of RNASET2 were clearly recognized by the anti-RNASET2 polyclonal antibody (see Figs. 4, 5, and Supplementary Fig. 2). Expression and Purification of RNASET2 Protein Lacking the TRAF2-Binding Motif We have recently demonstrated that the human RNASET2 gene acts as a tumor antagonizing gene in an ovarian carcinoma model. Importantly, we have also demonstrated that a catalytically impaired form of the protein, in which two key histidine residues strictly required for the enzymatic activity were replaced by phenylalanine (H65F/ H118F), still retained the ability to suppress tumor growth in vivo and recruit cells belonging to the monocyte/macrophage lineage [7]. However, since functional mapping data for the RNASET2 protein are still lacking, it would be of crucial importance to identify the

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Fig. 3 SEC and SDS-PAGE of delTRAF2bd_RNASET2-His. a Amino acid sequences of the mature forms of wild-type RNASET2 and its delTRAF2bd_RNASET2-His deletion mutant (lacking the TRAF2 binding motif PKQE) were aligned and numbered according to the primary sequence of the human preprotein (which carries a 24aa leader peptide, not reported here). Due to cloning procedures, the delTRAF2bd_RNASET2-His construct contains two novel amino acids (i.e., EF, gray lowercase) at the N-terminal end. The HA and 69 His tags at the C-terminus of this mutant are in bold and in a box, respectively. CASI and CASII sites are underlined with solid and

dashed lines, respectively. b The delTRAF2bd_RNASET2-His protein was isolated from P. pastoris conditioned medium by a combination of IMAC and negative-mode IEX. The purified mutant was analyzed by both SEC on a Superdex 75 (10 9 300 mm) column and SDS-PAGE on 4–12 % gradient gels. Shown are a representative UV chromatogram and gels run under reducing conditions, where proteins were revealed by either silver staining or western blotting using an anti-His monoclonal antibody (1 lg and 200 ng of total proteins/lane, respectively)

motifs/domains responsible for its tumor suppressive activity. The recent structural characterization of the human RNASET2 protein revealed a high homology with other well-characterized members of the T2 RNases family, with the exception of the C-terminal portion, where a putative TRAF2-binding motif (PKQE, residues 222–225 of the preprotein) has been predicted in silico for the human protein. Significantly, a physical and functional interaction between human RNASET2 and TRAF2 has also been reported recently [12].

In order to investigate the role of the putative TRAF2binding motif in the tumor suppressor activity of RNASET2, the availability of recombinant RNASET2 protein lacking this motif would provide a fundamental tool. To this end, we assembled a recombinant construct by inserting a cDNA fragment coding for a PKQE-deleted human RNASET2 protein in the pPICZaA expression vector (Fig. 3a), following previously described cloning procedures [9]. The PKQE-deleted RNASET2 recombinant protein was expressed in P. pastoris and purified from cell

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Fig. 4 IMAC of wt_RNASET2-His and wt_2tag_RNASET2-His. a Amino acid sequences of the mature forms of wt_RNASET2-His (carrying a single 69 His tag at the C-terminal end) and wt_2tag_RNASET2-His (carrying two 69 His tags at either end) were aligned with that of the wild-type RNASET2 protein. Numbering is based on the primary sequence of the human preprotein. Both wt_RNASET2-His and wt_2tag_RNASET2-His have two N-terminal novel amino acids (EF, gray lowercase). HA tags are marked in bold, and 69 His sequences are reported within boxes. CASI and CASII sites are underlined with solid and dashed lines, respectively. b wt_RNASET2-His and wt_2tag_RNASET2-His were purified from P. pastoris conditioned media using Ni–NTA agarose beads on gravity-flow columns. Unfractionated materials (input) and Ni–NTA

fractions (flow-through, FT, wash, and eluates) were normalized by volume and separated on 13 % Tris–glycine gels under denaturing and reducing conditions. Proteins were revealed by western blotting with a polyclonal anti-RNASET2 antibody and analyzed as described in Table 2. c The same conditioned media as in B were passed ¨ KTA Purifier through a HisTrap HP (1 ml) column online to an A FPLC system. Chromatograms obtained from wt_RNASET2-His and wt_2tag_RNASET2-His are reported as dashed (19 His Tag) and solid (29 His Tag) lines, respectively. The dotted line indicates concentration of the eluent buffer (%B). Fractions eluted with 20, 40, and 250 mM imidazole were normalized by volume and run on 4–12 % gradient gels under denaturing and reducing conditions. Representative silver-stained gels are shown in the inset

culture supernatants using the IMAC/IEX chromatography strategy described above (Table 1). The unbound fraction obtained from IEX was then analyzed by both SEC and SDS-PAGE, resulting in a discrete peak corresponding to highly pure PKQE-deleted RNASET2 recombinant protein (Fig. 3b). Also for this purified recombinant mutant RNASET2 protein, the level of endotoxin contamination proved to be under the threshold value of 0.125 EU/ml/ 100 lg proteins.

purification of recombinant His-tagged RNASET2, we planned to modify the protein sequence in order to improve its recovery rate. We therefore decided to assess the effects of the addition of a second 69 His tag at the N-terminus of the wild-type RNASET2 protein. To this end, starting from the recombinant construct pPICZaA-wtRNASET2 already available in our laboratory [9], we cloned a DNA sequence encoding a second 69 His tag downstream of the cleavage site of the secretion peptide, where the N-terminal aminoacid sequence of native RNASET2 starts (Fig. 4a). The P. pastoris X33 strain was transformed with the recombinant construct, and expression of double-tagged RNASET2 protein in selected clones was induced in small scale (50 ml of supernatant), including a single-tagged wtRNASET2-expressing clone as an internal control. The single clone displaying the highest expression levels of the double-tagged RNASET2 protein was identified by immunoblotting on culture supernatants. Western blot analysis showed that the overall expression levels of the double-tagged protein were comparable to the

Expression and Purification of Double 69 His-Tagged RNASET2 Protein Despite the optimization steps described above, recombinant single-tagged (69 His) RNASET2 still displayed a rather low affinity for the Ni2?-loaded stationary phases used in both gravity-flow and FPLC IMAC columns, thus leading to significant protein loss in the unbound fraction of IMAC even when the binding/capturing conditions were modified (Table 1). Being IMAC a mandatory step for the

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Mol Biotechnol Table 2 Comparison of IMAC purification of wt_RNASET2-His and wt_2tag_RNASET2-His from P. pastoris Fraction

Band density (AU)

Loaded volume (ll)

Total volume (ll)

Total density (AU)

Protein amount (lg)

Protein yield (%)

wt_RNASET2-His (11.7 mg/l)a Input

120,561

2.50

9,000

4.3E?08

584

100

FT Wash

38,937 3,005

2.50 0.56

9,000 2,000

1.4E?08 1.1E?07

189 15

32 2

Eluate

66,073

0.22

800

2.4E?08

320

55

100

wt_2tag_RNASET2-His (9.9 mg/l)a Input

80,616

2.50

9,000

2.9E?08

495

3,277

2.50

9,000

1.2E?07

20

4

Wash

0

0.56

2,000

0.0E?00

0

0

Eluate

79,209

0.22

800

2.9E?08

486

98

FT

a

Conditioned media (50 ml) were concentrated to a final volume of 9 ml (*5.6 fold) prior to IMAC. The values here reported refer to protein concentration in the non-concentrated media, as determined by densitometric analysis

expression levels of the single-tagged one (Fig. 4b, Input lane; Table 2). Both single- and double-tagged recombinant proteins were then purified from conditioned media using gravity-flow affinity chromatography, and fractions from IMAC were analyzed by SDS-PAGE followed by immunoblotting. As shown in Fig. 4b, a significant reduction of the RNASET2 signal in the flow-through fraction was observed for the double-tagged RNASET2 with respect to the single-tagged protein, suggesting that the issue of protein loss was properly addressed by this strategy. Moreover, the signal from double-tagged RNASET2 was below the detection limit in the wash fraction. A more rigorous analysis was performed by a densitometric quantification of the immunoblot signals followed by normalization of band density by loaded volume and total volume of fractions (Table 2). This allowed us to directly compare the relative protein yields, since the values obtained in this way were independent from protein concentration in the different preparations. As expected from the data reported above, the densitometric analysis showed that addition of a second 69 His tag at the N-terminus resulted in a consistent improvement of the overall relative yield of recombinant RNASET2, which was increased from 55 % (single tag) to 98 % (double tag). In order to further confirm these improvements in terms of recovery rate of the double-tagged RNASET2 recombinant protein, two batches of P. pastoris supernatants (500 ml) expressing either the single- or the double-tagged RNASET2 were purified by IMAC using HisTrap HP columns operated by an FPLC system. As shown in Fig. 4c (silver-stained gels in the figure inset), the total protein content in fractions eluted at low imidazole (20 and 40 mM) was significantly lowered for double-tagged RNASET2 protein with respect to the single-tagged one.

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Of note, these differences were not paralleled by a concomitant reduction of the corresponding chromatogram signals (Abs280nm at 20 and 40 mM imidazole). A reasonable explanation for this is that both peaks are mostly due to UV-absorbing components of the medium (e.g., peptides, amino acids, vitamins), which are clearly present in both the single- and double-tagged protein containing P. pastoris supernatants. As expected, protein recovery in the 250 mM imidazole eluted fraction turned out to be increased for the double-tagged protein, thus confirming our previous results from small-scale purification assays using gravity-flow affinity chromatography. Taken together, our results suggest that coupling the expression of the double-tagged recombinant RNASET2 protein in P. pastoris with the purification protocol described above (IMAC followed by negative-mode anion exchange chromatography) could represent the best approach to obtain highly pure endotoxin-free recombinant RNASET2 protein at high yields. Zymographic Analysis and Deglycosylation of RNASET2 Protein As described above, the PKQE-deleted RNASET2 protein would be a crucial tool in order to assess the functional properties of this TRAF2-binding motif. On the other hand, the expression of the double-tagged RNASET2 protein harbors a clear advantage in terms of recovery rate. However, the introduced modifications could alter the folding process of the recombinant protein and, more in general, affect its physical properties and functional features. We therefore set up a zymographic analysis in order to assess whether these modifications could influence the ribonuclease activity of the RNASET2 protein. To this end,

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Fig. 5 Zymographic analysis of the RNASET2 proteins. wt_RNASET2-His, wt_2tag_RNASET2-His, and delTRAF2bd_RNASET2His were run on 13 % Tris–glycine gels under denaturing/nonreducing conditions (600 ng of total proteins/lane). Proteins’ catalytic activity was assessed by zymography as described in ‘‘Materials and Methods’’ section. The H65F/H118F_RNASET2-His mutant (i.e., that is catalytically inactive) was used as a negative control (upper panel). Protein identity was verified by immunoblotting with a polyclonal anti-RNASET2 antibody (lower panel)

the whole set of recombinant RNASET2 proteins described above was run on RNA-containing polyacrylamide gels in denaturing/non-reducing conditions. After renaturation, a gel activity assay was performed. As shown in Fig. 5, upper panel, the expected bleaching pattern of toluidine staining (indicative of RNAse activity) was observed for both single- and double-tagged wild-type RNASET2 protein, as well as for the PKQE-deleted protein, thus, confirming the maintenance of the ribonuclease activity. The H65F/H118F_RNASET2-His, which is catalytically inactive, was used as an internal negative control and gave the expected non-bleaching pattern. Immunoblot analysis was also performed on these samples and confirmed that the bleached spots on the gel were indeed due to RNASET2 activity (Fig. 5, lower panel). These results demonstrated that the presence of the additional 69 His tag, as well as the deletion of the TRAF2 binding motif, does not impair the ribonuclease activity of the recombinant RNASET2 protein. Finally, we investigated the nature of the apparent differences in molecular weight observed for all forms of recombinant RNASET2 protein produced. In fact, the different batches of recombinant protein did not show the same migration pattern after SDS-PAGE, despite being all responsive to the anti-RNASET2 antibody. Although a

small batch-to-batch variation with the same heterologous expression system could be observed, the most significant differences emerged when comparing recombinant RNASET2 proteins produced in BEVS with those expressed in P. pastoris. In our previous work [9], we have already demonstrated that the expression of the RNASET2 protein in the P. pastoris system resulted in the production of both mildly and highly glycosylated forms of the protein, which were not expressed in BEVS. To verify that the observed differences reflect different patterns of protein glycosylation, we treated the whole set of recombinant RNASET2 proteins with PNGaseF enzyme after denaturation. The results clearly demonstrated that, once de-glycosylated, all the batches of RNASET2 protein migrate at the same molecular weight after SDS-PAGE separation and immunoblotting (supplementary Fig. 2). In this context, it is important to highlight that the migration pattern of the same batch of recombinant protein could differ slightly from one SDS-PAGE gel to another, solely due to an intrinsic limitation of this experimental procedure.

Discussion RNA degradation is a very ancient and highly conserved process. Indeed, all life forms are endowed with a complex set of ribonucleolytic enzymes, which play key roles in a wide range of biological processes. Besides the obvious role of clearing cellular RNA that is no longer required, RNases play other critical functions such as maturation of functional RNAs, control of endogenous gene expression by post-transcriptional RNA processing, and host defense against several pathogens [1]. Moreover, the pleiotropic function of these enzymes has been further demonstrated by the observation that a few RNases are able to play their biological roles independently of their ribonucleolytic activity [3]. In recent years, among the biological roles ascribed to ribonucleases, a growing interest has been focused on their oncosuppressive effect [13]. Indeed, several RNases belonging to both A and T2 families have been shown to display a powerful anticancer activity in both in vitro and in vivo experimental models. For instance, the RNase A family member ranpirnase (also known as onconase) has long been known to possess a strong cytotoxic activity for tumor cells and has been evaluated in clinical trials for the treatment of malignant mesotheliomas [14]. More recently, members of the more evolutionary conserved T2 family of secreted ribonucleases have also been reported to possess a strong oncosuppressive potential [3], and our group has recently reported such a role for RNASET2, the only human member of this protein family.

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Indeed, a strong oncosuppressive activity was reported for RNASET2 in an in vivo human ovarian cancer model based on xenograft assays [7, 8]. Significantly, the catalytic activity of the RNASET2 protein was not required for its oncosuppressive role, as reported for other members of this family [15, 16]. By contrast, secretion of RNASET2 by cancer cells turned out to be strictly required for tumor suppression, pointing to a non-cell autonomous role for this protein (Lualdi et al., manuscript submitted). Indeed, extracellular RNASET2 released by cancer cells in vivo was shown to trigger the massive recruitment of cells from the monocyte/macrophage lineage in the tumor mass, where they were functionally involved in tumor shrinkage [7, 8]. The RNASET2 protein is produced by cells in three different forms: a full-length 36 kDa form that is secreted by cells and two intracellular forms (31 and 27 kDa) which originate from proteolytic cleavage at the C-terminus of the full-length protein [4]. While a consistent evolutionary conservation is observed among the catalytic core structures of T2 RNases, the C-terminal portion of human RNASET2 (which is retained in the secreted protein form) differs significantly from that encoded by other structurally characterized orthologs [5]. Interestingly, recombinant RNASET2 protein lacking the entire C-terminal portion of the protein (aa 214–256 of the preprotein) turned out to be expressed but not secreted by Pichia pastoris cells (Lualdi, unpublished data), suggesting that this region might play a crucial role also for proper protein folding/stability and possibly for its engagement in the secretory pathway. Similar results were also obtained in mammalian cells, where different deletion forms of the protein could be expressed but not secreted by human ovarian cancer cells (Lualdi, unpublished data). Furthermore, a putative TRAF2-binding motif (PKQE) has also been predicted in silico in the C-terminal portion of human RNASET2, and physical interaction between RNASET2 and TRAF2 proteins has been very recently reported [12]. Since extracellular RNASET2 is strictly required for tumor suppression in vivo (Lualdi et al., manuscript submitted), it is tempting to speculate that the C-terminal portion of RNASET2 (which is retained in the secreted 36 kDa form and carries the putative TRAF2-binding motif) might harbor some functional motifs/domains involved in the catalysis-independent oncosuppressive role of RNASET2. In keeping with this hypothesis, we have recently demonstrated that both wild-type and catalytically impaired RNASET2 proteins are endowed with a strong chemotactic activity when exogenously provided to human cells belonging from the monocyte/macrophage lineage [7, 8]. Since TRAF-2 plays a key role in innate immune response, the observed chemotactic properties of recombinant RNASET2 on monocyte/macrophages, coupled to

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the recently reported interaction between RNASET2 and TRAF-2 proteins, strongly suggests that the interaction between these two proteins might be functionally involved in RNASET2-mediated activation of innate immunity. However, to further define the role of extracellular RNASET2 in these biological responses, the availability of highly pure, endotoxin-free recombinant RNASET2 protein represents an essential step. In this work, we report a significant improvement in purification protocols for recombinant human RNASET2 protein expressed in both BEVS and P. pastoris heterologous expression systems. Indeed, we provide here experimental evidence that: (1) 69 His-tagged RNASET2 protein can be purified from both BEVS and P. pastoris supernatants by coupling gravity-flow IMAC to a SECFPLC polishing step, leading to homogeneous preparations although with quite low recovery rates and persistent endotoxin contamination; (2) IMAC-FPLC coupled to negative-mode anion exchange chromatography can bring the levels of LPS in recombinant RNASET2 protein preparations from P. pastoris below the threshold set and improve the overall protein yield (as compared to IMAC/ SEC), however, performance of IMAC remains poor; (3) the addition of a second 69 His tag at the N-terminus of the protein greatly increases the recovery rate of the recombinant RNASET2 protein expressed and purified from P. pastoris using IMAC; (4) a PKQE-deleted recombinant RNASET2 protein can be efficiently expressed and purified from P. pastoris supernatants; and (5) finally, all these purification approaches do not impair the structure and function of the recombinant protein, as assessed by catalytic activity assays. Collectively, these results will be of great practical value for future in vitro assays aimed at fully characterizing the functional properties of the RNASET2 protein. Indeed, migration assays on monocytes from buffy coats will be performed using endotoxin-free PKQE-deleted recombinant RNASET2 protein as a chemoattractant with respect to the wild-type protein, in order to assess the functional role of this four amino acids motif in recruitment and possibly activation of monocytes/macrophages in vivo. Moreover, treatment of cells belonging to the monocyte/macrophage lineage with both forms of recombinant RNASET2 protein will allow us to assess the occurrence of alterations in the actin cytoskeleton pattern of these cells. In fact, we have recently unveiled a role for RNASET2 protein in the reorganization of the actin cytoskeleton in human ovarian tumor cells, with important consequences on migration and invasion potentials (Lualdi et al., manuscript submitted). Interestingly, such a role is not unique to human RNASET2, since the T2 ribonuclease ortholog Omega-1 secreted from Schistosoma mansoni eggs exerts its role in conditioning dendritic cells to promote Th2

Mol Biotechnol

differentiation by inducing profound changes in the actin cytoskeleton of these cells [17, 18]. Finally, the use of the recombinant RNASET2 protein will be instrumental in the search for a putative RNASET2 receptor molecule on both tumor and monocyte/macrophage cells. In this context, it has been recently reported that omega-1 protein is bound and internalized via its glycans by the mannose receptor in dendritic cells and subsequently impairs protein synthesis by degrading both rRNAs and mRNAs [19]. Being human RNASET2 protein also glycosylated, one of our next aims would be the investigation of a similar mechanism in human macrophage cells. In this regard, it is worth pointing out that the purification approaches described here allow separation of RNASET2 expressed in P. pastoris from its highly glycosylated forms (that are not present in the human molecule), thus leading to a glycosylated recombinant product that is more amenable to in vitro and in vivo investigations on the oncosuppressive properties of this peculiar RNase.

8.

9.

10.

11.

12. Acknowledgments F. A. was supported by FAR Insubria Univer˚ bo Akademi sity Academic Fund. C. Lindqvist was supported by A University Foundation. A. I. is grateful for support from the European Research Council and the Associazione Italiana per la Ricerca sul Cancro.

13.

14.

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New Strategies for Expression and Purification of Recombinant Human RNASET2 Protein in Pichia pastoris.

Ribonucleases form a large family of enzymes involved in RNA metabolism and are endowed with a broad range of biological functions. Among the differen...
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