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— Cell culture protein surface coating is a procedure in which the cell culture surfaces are coated with proteins or extracellular matrix (ECM) components to enhance the adhesion and proliferation of the cells during in vitro isolation and cultivation. Proteins that are used in coating cells are collagen, fibronectin, vitronectin, laminin, and osteopontin, which are either animal derived, plant derived, synthetic, or human derived. Protein surface coating facilitates the growth of various types of cells such as epithelial, leukocytes, muscle cells, neurons, chinese hamster ovary (CHO) cell lines, fibroblasts, and neurons. Publisher's analysts forecast the global cell culture protein surface coating market to grow at a CAGR of 13.20% during the period 2017-2021. Covered in this report The report covers the present scenario and the growth prospects of the global cell culture protein surface coating market for 2017-2021. To calculate the market size, the report presents a detailed picture of the market by the way of study, synthesis, and summation of data from multiple sources. The market is divided into the following segments based on geography: - Americas - APAC - EMEA Publisher's report, Global Cell Culture Protein Surface Coating Market 2017-2021, has been prepared based on an in-depth market analysis with inputs from industry experts. The report covers the market landscape and its growth prospects over the coming years. The report also includes a discussion of the key vendors operating in this market. Get Sample of the Report at: http://www.reportsweb.com/inquiry&RW0001713445/sample . Other prominent vendors - Abcam - Agilent Technologies - AM-Pharma - BioLamina - BioMedTech Laboratories - Bio-Techne - BioTime - Caladrius Biosciences - Cedarlane Laboratories - Cell Guidance Systems - CellSystems Biotechnologie Vertrieb - Cellular Dynamics International - Cytoskeleton - Full Moon BioSystems - Greiner Bio-One - GlaxoSmithKline - Histocell - Japan Regenerative Medicine - KANGSTEM BIOTECH - Mesoblast - neuVitro - Orla Protein Technologies - Pall - PerkinElmer - PROGEN Biotechnik - PromoCell - RayBiotech - Sartorius Stedim Biotech - SouthernBiotech - Taiwan Bio Therapeutics - Takeda Pharmaceutical Company - Teva Pharmaceutical Industries - Trevigen - TWO CELLS - U.S. Stem Cell - Viogene Market driver - Government and research centers to promote research activities. - For a full, detailed list, view our report Market challenge - Ethical concerns over usage of animal-derived protein coating material. - For a full, detailed list, view our report Market trend - Increasing preference for 3D cell cultures over 2D cell cultures. - For a full, detailed list, view our report PART 01: Executive summary PART 02: Scope of the report PART 03: Market research methodology PART 04: Introduction PART 05: An overview: Cell culture surfaces PART 06: Market landscape PART 07: Market segmentation by product type PART 08: Geographical segmentation PART 09: Decision framework PART 10: Drivers and challenges PART 11: Market trends PART 12: Vendor landscape PART 13: Key vendor analysis PART 14: Appendix For more information, please visit http://www.reportsweb.com/global-cell-culture-protein-surface-coating-market-2017-2021

3 Résultat net courant = résultat net après les participations ne donnant pas le contrôle, corrigé des éléments non récurrents, hors Amortissement et basé sur le résultat financier et le taux d’impôt normalisés. Sartorius Stedim Biotech est l'un des principaux fournisseurs internationaux de produits et services dans l'industrie biopharmaceutique, destinés au développement et à la fabrication de produits pharmaceutiques en toute sécurité et efficacité. En tant que fournisseur de solutions intégrées, le portefeuille de Sartorius Stedim Biotech couvre presque toutes les étapes de la production biopharmaceutique. Avec sa forte concentration sur les technologies à usage unique et les services à valeur ajoutée, Sartorius Stedim Biotech est au cœur de la mutation technologique fulgurante de son secteur d'activité. Basée à Aubagne en France, Sartorius Stedim Biotech est cotée sur Euronext à la Bourse de Paris. Dotée de ses propres sites de production et de R&D en Europe, en Amérique du Nord et en Asie, ainsi que d'un réseau de distribution international, Sartorius Stedim Biotech est présente dans le monde entier. Le groupe Sartorius Stedim Biotech a employé environ 4 700 personnes et a réalisé un chiffre d'affaires de 1 052 millions d'euros sur l'exercice 2016.

This report studies the global High Throughput Process Development market, analyzes and researches the High Throughput Process Development development status and forecast in United States, EU, Japan, China, India and Southeast Asia. This report focuses on the top players in global market, like Market segment by Type, High Throughput Process Development can be split into Instrument Software and Service Market segment by Application, High Throughput Process Development can be split into Monoclonal Antibodies Recombinant Insulin Global High Throughput Process Development Market Size, Status and Forecast 2022 1 Industry Overview of High Throughput Process Development 1.1 High Throughput Process Development Market Overview 1.1.1 High Throughput Process Development Product Scope 1.1.2 Market Status and Outlook 1.2 Global High Throughput Process Development Market Size and Analysis by Regions 1.2.1 United States 1.2.2 EU 1.2.3 Japan 1.2.4 China 1.2.5 India 1.2.6 Southeast Asia 1.3 High Throughput Process Development Market by Type 1.3.1 Instrument 1.3.2 Software and Service 1.4 High Throughput Process Development Market by End Users/Application 1.4.1 Monoclonal Antibodies 1.4.2 Recombinant Insulin 2 Global High Throughput Process Development Competition Analysis by Players 2.1 High Throughput Process Development Market Size (Value) by Players (2016 and 2017) 2.2 Competitive Status and Trend 2.2.1 Market Concentration Rate 2.2.2 Product/Service Differences 2.2.3 New Entrants 2.2.4 The Technology Trends in Future 3 Company (Top Players) Profiles 3.1 Danaher Corporation 3.1.1 Company Profile 3.1.2 Main Business/Business Overview 3.1.3 Products, Services and Solutions 3.1.4 High Throughput Process Development Revenue (Value) (2012-2017) 3.1.5 Recent Developments 3.2 GE Healthcare 3.2.1 Company Profile 3.2.2 Main Business/Business Overview 3.2.3 Products, Services and Solutions 3.2.4 High Throughput Process Development Revenue (Value) (2012-2017) 3.2.5 Recent Developments 3.3 Agilent Technologies 3.3.1 Company Profile 3.3.2 Main Business/Business Overview 3.3.3 Products, Services and Solutions 3.3.4 High Throughput Process Development Revenue (Value) (2012-2017) 3.3.5 Recent Developments 3.4 Merck Millipore 3.4.1 Company Profile 3.4.2 Main Business/Business Overview 3.4.3 Products, Services and Solutions 3.4.4 High Throughput Process Development Revenue (Value) (2012-2017) 3.4.5 Recent Developments 3.5 Thermo Fisher Scientific 3.5.1 Company Profile 3.5.2 Main Business/Business Overview 3.5.3 Products, Services and Solutions 3.5.4 High Throughput Process Development Revenue (Value) (2012-2017) 3.5.5 Recent Developments 3.6 Bio-Rad Laboratories 3.6.1 Company Profile 3.6.2 Main Business/Business Overview 3.6.3 Products, Services and Solutions 3.6.4 High Throughput Process Development Revenue (Value) (2012-2017) 3.6.5 Recent Developments 3.7 Eppendorf AG 3.7.1 Company Profile 3.7.2 Main Business/Business Overview 3.7.3 Products, Services and Solutions 3.7.4 High Throughput Process Development Revenue (Value) (2012-2017) 3.7.5 Recent Developments 3.8 Perkinelmer, Inc 3.8.1 Company Profile 3.8.2 Main Business/Business Overview 3.8.3 Products, Services and Solutions 3.8.4 High Throughput Process Development Revenue (Value) (2012-2017) 3.8.5 Recent Developments 3.9 Sartorius Stedim Biotech 3.9.1 Company Profile 3.9.2 Main Business/Business Overview 3.9.3 Products, Services and Solutions 3.9.4 High Throughput Process Development Revenue (Value) (2012-2017) 3.9.5 Recent Developments 3.10 Tecan Group Ltd. 3.10.1 Company Profile 3.10.2 Main Business/Business Overview 3.10.3 Products, Services and Solutions 3.10.4 High Throughput Process Development Revenue (Value) (2012-2017) 3.10.5 Recent Developments 4 Global High Throughput Process Development Market Size by Type and Application (2012-2017) 4.1 Global High Throughput Process Development Market Size by Type (2012-2017) 4.2 Global High Throughput Process Development Market Size by Application (2012-2017) 4.3 Potential Application of High Throughput Process Development in Future 4.4 Top Consumer/End Users of High Throughput Process Development For more information, please visit https://www.wiseguyreports.com/sample-request/1206536-global-high-throughput-process-development-market-size-status-and-forecast-2022

No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment except when counting VSV-G spikes (Extended Data Fig. 7b). I3-01–Myc and EPN-01* mammalian expression constructs were generated by PCR amplification of the coding sequences from a pET29b-EPN-01* expression vector and transferred into a CMV-based mammalian expression vector (pCMV, DNASU ID: EvNO00601609 (ref. 31)) using the NotI and XhoI restriction sites. Plasmids for mammalian cell expression of EPNs -01 through -51 were constructed and inserted into pCMV using the KpnI and XhoI restriction sites by Gibson Assembly32 using synthetic DNA (Gen9). The E. coli expression plasmid for EPN-01* was constructed by adding the N- and C-terminal functional elements to the I3-01 sequence9 by PCR and inserting it by Gibson Assembly into pET29b digested with NdeI and XhoI. Mutations were introduced by round-the-horn site-directed mutagenesis or PCR amplification followed by Gibson Assembly as indicated in Supplementary Table 3. All constructs were verified by sequencing. A comprehensive list of all plasmids and coding sequences sources is provided in Supplementary Table 3. All of the plasmids have been submitted to the Addgene repository ( https://www.addgene.org/). A comprehensive list of all antibodies, sources and dilutions is provided in Supplementary Table 4. Expression plasmids were transformed into BL21(DE3) E. coli cells, and cells were grown in LB medium supplemented with 50 mg l−1 kanamycin (Sigma) at 37 °C to an OD of 0.8. Protein expression was induced by addition of 0.5 mM isopropyl-thio-β-d-galactopyranoside (Sigma) and allowed to proceed for 3 h at 37 °C before cells were harvested by centrifugation. For the EPN-01* protein shown in Extended Data Figs 2c and 3a, b cells from a 1 l expression culture were lysed by sonication in 20 ml of 50 mM Tris pH 8, 250 mM NaCl, 20 mM imidazole, 2.5 mM MgCl , 0.5 mM CaCl , 1 mM DTT, 1 mM phenylmethanesulfonyl fluoride (PMSF) supplemented with 20 mg DNase (Sigma) and 2 mg of RNase (Qiagen), and the lysates were clarified by centrifugation for 25 min at 51,000g, 4 °C. Ammonium sulphate was added to the clarified lysate to 60% saturation, incubated at room temperature for 15 min, and the precipitate pelleted by centrifugation for 15 min at 51,000g, 4 °C. The pelleted protein was resuspended in 20 ml of 25 mM Tris pH 8, 150 mM NaCl, 5 mM EDTA, 1 mM DTT and heated for 10 min at 75 °C. The solution was clarified by centrifugation for 15 min at 51,000g, 4 °C, filtered with a 0.45 μm filter (EMD Millipore), and concentrated using a Centricon concentrator (EMD Millipore). The protein was then purified using a Superose 6 10/300GL column in the same buffer, the fractions pertaining to the nanocage peak centred around 12 ml were pooled and concentrated, and the protein refractionated using the Superose 6 10/300GL column equilibrated in 25 mM Tris pH 8, 150 mM NaCl, 5 mM EDTA supplemented with 0.75% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Nanocage peak fractions were again pooled and concentrated, and protein concentration determined using the BCA assay (ThermoFisher). The proteins shown in Extended Data Figs 2a and 1b were purified using a combination of immobilized metal affinity chromatography (IMAC) and size-exclusion chromatography using a Superose 6 10/300 GL column. E. coli cells were lysed by sonication in 25 mM TRIS pH 8.0, 250 mM NaCl, 1 mM DTT, 20 mM imidazole supplemented with 1 mM phenylmethanesulfonyl fluoride, and the lysates were cleared by centrifugation for 25 min at 51,000g, 4 °C and filtered through 0.22 μm filters (Millipore). The proteins were purified from the filtered supernatants by IMAC via linear gradient elution from HisTrap HP columns (GE Healthcare) using 25 mM TRIS pH 8.0, 250 mM NaCl, 1 mM DTT, 20 mM imidazole as running/wash buffer and 25 mM TRIS pH 8.0, 250 mM NaCl, 1 mM DTT, 500 mM imidazole as elution buffer. Elution fractions containing pure proteins of interest were pooled, concentrated using centrifugal filters (Sartorius Stedim Biotech), and further purified on a Superose 6 10/300 gel filtration column (GE Healthcare) using 25 mM TRIS pH 8.0, 150 mM NaCl, 1 mM DTT as running buffer. For the negative stain electron microscopy image shown in Extended Data Fig. 2c, 6 μl of purified EPN-01* at 0.075 mg ml−1 were applied to glow discharged, carbon-coated 400-mesh copper grids (Ted Pella), washed with Milli-Q water and stained with 0.75% uranyl formate. Grids were visualized for assembly validation and optimized for data collection. Screening and sample optimization was performed on a 100 kV Morgagni M268 transmission electron microscope (FEI) equipped with an Orius charge-coupled device (CCD) camera (Gatan). The final image was recorded on a 120 kV Tecnai G2 Spirit transmission electron microscope (FEI) using an Ultrascan 4000 4k × 4k CCD camera (Gatan) at 52,000× magnification at the specimen level. HeLa and HEK293T (293T) cells were obtained from ATCC and cultured in D-MEM (ThermoFisher) containing 10% FBS, penicillin (100 U ml−1) and streptomycin (0.1 mg ml−1), at 37 °C and 5% CO . Expi293F (293F) cells, used to survey different EPN constructs, were obtained from ThermoFisher and cultured in 293F Expression Medium (ThermoFisher) containing penicillin (100 U ml−1) and streptomycin (0.1 mg ml−1), at 37 °C and 5% CO while shaking at 125 r.p.m. Cells were tested for mycoplasma contamination every 3 months using the MycoAlert Mycoplasma Detection Kit (Lonza). To assay EPN release as shown in Figs 1, 4 and Extended Data Fig. 1, 8 × 105 293T cells were seeded in 6-well plates 24 h before transfection. Cells were transfected with 2 μg of plasmid DNA expressing the I3-01- or O3-33-based EPN constructs, or co-transfected with 1 μg of plasmids expressing I3-01-based constructs and 1 μg of either pEGFP-VPS4A(E228Q) or pEGFP-C1 (Clontech), using the polyethyleneimine (PEI, Polysciences, 3 μl of PEI per μg DNA) method. The medium was replaced with 1 ml growth medium 5 h later. Cells and culture supernatants were harvested 24 h post transfection. Released EPN assemblies were collected from the culture supernatants by centrifugation through a 200 μl 20% sucrose cushion for 90 min at 21,000g, 4 °C, and denatured by adding 50 μl 1 × Laemmli buffer and boiling for 5 min. Cells were lysed for 5 min on ice in 200 μl cold lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, protease inhibitors). Lysates were clarified by centrifugation for 5 min, 16,000g, 4 °C. The Triton-soluble fraction was treated with 200 μl 2 × Laemmli buffer supplemented with 10% 2-mercaptoethanol (Sigma) and boiled for 5 min. Triton-insoluble material was solubilized in 200 μl 2 × Laemmli buffer by boiling for 10 min. Samples containing EPN-51 and mutants thereof were incubated for 30 min at 40 °C instead of boiling because the protein aggregated at high temperatures. Benzonase (Sigma, 0.5 μl per sample) was added to remove nucleic acids from the Triton-insoluble fractions. The Triton-soluble and -insoluble cellular fractions, and the released EPN complexes, were separated by 12.5% SDS–PAGE, transferred onto PVDF membranes (or nitrocellulose membranes in case of EPN-51 constructs), and probed with antibodies against the Myc epitope (primary antibodies and dilutions are provided in Supplementary Table 4). GAPDH was used as a loading control. Bands were visualized by probing the membrane with fluorescently labelled secondary antibodies (Li-Cor Biosciences) and scanning with an Odyssey Imager (Li-Cor Biosciences). Levels of expressed and released Myc-tagged EPN proteins were quantified by western blot densitometry with ImageJ33 using standard curves generated with known quantities of recombinant EPN-01* protein produced in E. coli. Release efficiencies are reported as the percentage of EPN protein pelleted from the supernatant versus the total protein in cells and pelleted supernatant. All experiments were repeated independently at least twice. Standard deviations shown in all figures were calculated from three technical repeats (three transfections in parallel) of each experiment. Released EPNs used in immunoprecipitation and cryo-EM studies were purified from culture supernatants of 293T cells (2 × 106 per 10 cm plate, 7 plates per specimen for the experiments shown in Fig. 2b and Extended Data Fig. 3d, 36 plates for the experiments shown in Fig. 2c, e and Extended Data Fig. 3a, seeded 24 h before transfection) after transient transfection with plasmids encoding EPN-01 or EPN-01* (12 μg per plate) using the calcium phosphate method (Clontech). Transfected cells were incubated overnight and the media was replaced with exosome production media (D-MEM supplemented with 10% FBS, depleted of contaminating extracellular particles by centrifugation overnight at 100,000g at 4 °C and subsequently filtered through a 0.22 μm filter)34. Cells were grown for an additional 24 h and extracellular EPN assemblies were purified by a series of filtering and centrifugation steps (adapted from ref. 34). In brief, cell debris was removed by centrifugation of the supernatant at 1,000g for 5 min followed by filtration through a 0.22 μm filter (EMD Millipore). EPN assemblies were collected by centrifugation at 100,000g in an SW32Ti (BeckmanCoulter) at 4 °C for 1 h. Pellets were resuspended in PBS and pooled in one tube (SW41 rotor, BeckmanCoulter). PBS was added to fill the tube completely and EPN assemblies were collected by centrifugation at 100,000g at 4 °C for 1 h. Pellets were resuspended in 1 ml of PBS and concentrated by centrifugation at 100,000g at 4 °C for 1 h in an OptimaMAX-E (BeckmanCoulter) bench-top ultracentrifuge using a TLS-55 rotor. EPNs were quantified by western blotting as described above. Typical yields were 2–8 μg EPN-01 and EPN-01* proteins from 36 × 10 cm dishes. EPNs for protease protection and aldolase activity assays as shown in Fig. 2a and Extended Data Figs 3b and 8 were prepared as follows. On the day of transfection, 293F cell count and viability were determined using trypan blue solution in a haemocytometer. The cells were plated in 1 ml volumes at 2.5 × 106 cells per ml on non-TC treated 12-well plates (Corning). The cells were transfected with 1 μg of plasmid DNA using Expifectamine transfection reagent (ThermoFisher) following the manufacturer’s instructions. A cocktail of Expifectamine 293F Transfection Enhancer1 and Enhancer2 (ThermoFisher) was added to each well following manufacturer’s instructions 18 h after transfection. Cells and cultured supernatants were collected 44 h post-transfection and separated by centrifugation for 5 min at 1,000g, 4 °C. The culture supernatants were filtered through a 0.45 μm filter (EMD Millipore) into a 1.5 ml microfuge tube. Released EPN assemblies were collected from the culture supernatants by centrifugation through 200 μl of a 20% sucrose cushion for 120 min at 21,000g, 4 °C and resuspended in PBS. Purified EPNs were resuspended and incubated under three different conditions, 10 μl each: untreated EPN, EPN + 0.05 mg ml−1 trypsin, and EPN + 0.05 mg ml−1 trypsin + 1% Triton X-100. Samples were incubated for 30 min at 25 °C and then 1 mM PMSF was added and incubated for 10 min at 25 °C to inactivate trypsin. Samples were denatured by boiling for 10 min in 4 × Laemmli buffer supplemented with 5% 2-mercaptoethanol (except samples with the O3-33 domain, which were not boiled). All fractions were separated by SDS–PAGE, transferred onto nitrocellulose membranes, and analysed by western blot using an anti-Myc antibody (Supplementary Table 4). Western blots were imaged using HRP-conjugated secondary antibodies (Cell Signaling Technology) and Clarity Western ECL Blotting Substrate (Bio-Rad). At least three biological replicates of the protease protection assay (independent transfections or batches of E. coli purified EPN-01*) were performed for each construct described in Extended Data Fig. 8 and Supplementary Table 1. The 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase activity of the I3-01 domain was monitored using a l-lactic acid dehydrogenase (LDH)-coupled assay35. 95-μl samples of assay solutions containing 25 mM HEPES pH 7.0, 20 mM NaCl, 0.1 mM NADH, 0.11 U μl−1 LDH, 1 mM KDPG, and either including or omitting 1% Triton X-100, were mixed with 5 μl of resuspended EPNs. Loss of absorbance at 339 nm owing to oxidation of NADH was monitored using a SpectraMax M3 plate reader. At least three biological replicates of the aldolase activity assay (independent transfections or batches of E. coli purified EPN-01*) were performed for each construct described in Extended Data Fig. 8 and Supplementary Table 1. A total of 1 μg of purified EPN-01 expressed in either bacteria or harvested from 293T supernatants was incubated in 250 μl PBS buffer containing either 0.1% or 0.5% CHAPS detergent for 20 min. EPN-01 assemblies were immunoprecipitated by addition of 30 μl of the indicated antibodies coupled to agarose resin and incubated for 14 h at 4 °C on a rotating shaker. Antibody-bound resins were: anti-rabbit-IgG-Agarose (Sigma) and anti-c-Myc-Agarose (Sigma). Resins were washed six times at 4 °C with 1 ml PBS/0.1% CHAPS buffers and resuspended in 250 μl in 1 × Laemmli buffer containing 10% 2-mercaptoethanol, boiled for 5 min, and analysed by western blotting. To prepare samples for cryo-EM tomography, 3 μl of purified EPNs in PBS (50 ng EPN-01 per μl as determined by western blotting versus a standard curve) were mixed with 3 μl of BSA-coated gold fiducials (10 nm size, Electron Microscopy Sciences). 3.5 μl of the suspension were applied to a glow-discharged R2/2 holey carbon coated EM grid (Quantifoil) within the environmental chamber of a Vitrobot (FEI) maintained at 4 °C, 80% relative humidity. Excess liquid was blotted for 7.5 s (0 mm offset) from the grids with filter paper (Whatman) before plunge freezing in liquid ethane. Cryo-grids were placed in a Gatan 626 cryoholder (Gatan) and imaged in a 200 kV Tecnai F20 microscope (FEI) equipped with a K2 summit direct electron detector (Gatan). Tilt series were recorded bidirectionally starting from 0° to ± ~60° with a 1° step size at a magnification of 22,500× and a defocus of −8 μm (total dose per specimen, ~300 e− Å−2) using low-dose mode in SerialEM36. Tomograms were generated using the IMOD software package37. Image stacks were aligned, binned by 4 and gold particles were erased using findbeads3d within IMOD. Aligned image stacks were Fourier filtered (cut-off 0.25, σ = 0.08) and tomographic reconstructions were performed using the simultaneous reconstruction technique (SIRT). Noise reduction was performed with the nonlinear anisotropic diffusion (NAD) method in IMOD37, using a K value of 0.04 with 12 iterations. Segmentation and isosurface rendering was done using Amira (Version 4.1.2, FEI). Individual particles that could be completely traced along slices though the z axis were manually identified and surrounded by a mask. The space inside the mask was then segmented and an isosurface was generated. Video segments were created in Amira and ffmpeg ( http://ffmpeg.org) was used to combine segments and generate the H.264 encoded Supplementary Video 1. 4 μg of EPNs in PBS purified as described in EPN purification were incubated with 0.75% CHAPS for 20 min at 4 °C (20 μl total volume). 3.5 μl samples were placed on glow discharged R2/2 holey carbon grids (Quantifoil) within the Vitrobot environmental chamber (maintained at 4 °C, 80% relative humidity), blotted for 11 s (0 mm offset) with filter paper, and plunge frozen in liquid ethane. Cryo-grids were imaged with a TF20 microscope operated at 200 kV (42,000× magnification and −0.7 to −3.3 μm defocus). Images were recorded on a Gatan K2 Summit direct electron detector. SerialEM36 was used to facilitate low-dose imaging and semi-automated data collection, and 60 frames were recorded of each view in super-resolution mode. Frames were aligned and summed by using MotionCor238. Three-dimensional images were reconstructed via routines implemented in the package SCIPION39. Contrast transfer function (CTF) parameters were determined using CTFFIND4 (ref. 40). Particle images were selected and extracted using Xmipp41, 42. Non-dose-weighted image sums from MotionCor2 were used for CTF determination and particle picking, with dose-weighted image sums used for all other steps. Extracted two-dimensional particle images were processed and then classified with the RELION software package43. Suitable two-dimensional class averages were used to determine an ab initio 3D model via the program RANSAC44. This model was then used as the starting model for 3D image reconstruction via RELION with icosahedral symmetry applied during the 3D reconstruction calculations. The resulting 3D map was masked and a B-factor was applied (post-processing) via an automated procedure in RELION43. The final map was constructed from 8,573 particles, and the resolution was determined to be 5.7 Å by the gold standard 0.143 criterion (Extended Data Fig. 4c). The design model9 was rigid-body fit into the completed 3D density, using UCSF Chimera45 to perform the fit and generate Supplementary Video 2. One additional residue (Lys2 of I3-01) was built into the density at the N-terminal end of the first helix of the I3-01 construct, and no additional density was visible for any of the remaining sequences outside the I3-01 domain. The resolution of the model/map fit was 7.1 Å (Fourier cross resolution 0.378 criterion46, see Extended Data Fig. 4c). For immunofluorescence imaging shown in Extended Data Fig. 5, 2 × 105 HeLa cells were seeded onto coverslips in 12-well plates and transfected with plasmids encoding EPN proteins the next day. 24 h post-transfection, cells were fixed with 3.5% paraformaldehyde in PBS for 10 min at room temperature, washed twice in PBS, blocked and permeabilized in a block/perm solution (0.1% Triton X-100, 3% BSA in PBS) for 10 min before primary anti-Myc antibodies were added (1 μg ml−1 in block/perm solution) and incubated for 1 h at room temperature. Cells were washed three times for 10 min in wash buffer (0.1% Triton X-100 in PBS), and Alexa 488-labelled secondary anti-mouse IgG antibody was added (ThermoFisher, 2 μg ml−1 in block/perm solution) and incubated for 1 h at room temperature. Nuclei and actin were stained by incubating cells with Hoechst 33342 (ThermoFisher, 1:10,000) and Phalloidin 647 (ThermoFisher, 1:40) in block/perm solution for 15 min. Cells were washed four times in wash buffer, and twice in PBS before mounting onto glass coverslips (Flouromount-G, Southern Biotech). Confocal immunofluorescence images were acquired using NIS Elements software on a Nikon A1 microscope. Final images were prepared in Image J (FIJI)33. For immunogold labelling experiments shown in Extended Data Fig. 6, 8 × 105 293T cells were seeded in 6-well plates 24 h before transfection (2 wells per sample). Cells were transfected with 2.5 μg of plasmid DNA expressing the EPN construct using Lipofectamine 2000 (ThermoFisher) following the manufacturer’s instructions. 24 h post-transfection, the media was removed, cells were knocked loose and washed off the plate in fixative (2% PFA/0.1% glutaraldehyde in PBS), transferred to 1.5-ml test tubes and incubated on a rocker for 16 h at 4 °C. Cells were then pelleted for 4 min at 16,000g and washed four times for 5 min in 1 ml PBS and twice for 5 min in 1 ml water on a rocker. Cell pellets were stained with 50 μl 2% uranyl acetate for 30 min and washed again three times for 5 min in 1 ml water. Samples were dehydrated in 1 ml of a graded series of ethanol in water (3 × 70% ethanol, 3 × 95% ethanol, 3 × 100% ethanol, 5 min each) and incubated 16 h in 1 ml of a 1:1 mixture ethanol:LR White (Sigma-Aldrich) at room temperature. Samples were then infiltrated by two 6 h incubations in 1 ml 100% LR White, and the resin was polymerized at 50 °C overnight. Thin sections (80 nm, cut by a diamond knife (Diatome) in a Leica EM UC6 ultratome (Leica)) were mounted onto support specimen nickel grids (Electron Microscopy Sciences). For immunogold labelling, grids were hydrated on 100-μl drops of PBS for 10 min, and reactive aldehydes were then deactivated by incubating grids on 100-μl drops of 50 mM glycine in PBS for 10 min. Grids were blocked for 1 h on 40-μl drops of blocking solution (5% BSA in PBS) and then incubated with primary anti-Myc antibodies (40-μl drops, 2 μg ml−1 in blocking solution) overnight at 4 °C in a moist chamber. The next day, grids were washed three times for 5 min on 100-μl drops of blocking solution and probed on 40-μl drops of secondary anti-mouse IgG antibody labelled with 10-nm gold particles (Ted Pella, 1:100 in blocking solution) for 2 h at room temperature. Grids were then washed three times for 5 min in blocking solution and three times for 5 min in water, dried, and viewed on a JEOL JEM1400 electron microscope at an accelerating voltage of 120 kV. To quantify immunogold staining, a region of interest was defined in ImageJ and particles were counted inside the defined area using the particle analyse function (see Extended Data Fig. 6 a–c, right panels)33. The ability of EPNs to deliver packaged enzymes into the cytoplasms of recipient cells, as shown in Fig. 3 and Extended Data Fig. 7, was evaluated using a modified version of the β-lactamase (BlaM) assay described previously15. 5 μg of a plasmid expressing an N-terminally Myc-tagged chimaeric BlaM–Vpr fusion protein was co-transfected with 9 μg EPN-encoding plasmids and 1 μg of a plasmid encoding a C-terminally Myc-tagged VSV-G (Supplementary Table 3), in 10-cm plates using the Lipofectamine (ThermoFisher) method. Transfection medium was replaced with 10 ml growth medium 5 h post-transfection. EPNs containing VSV-G and BlaM–Vpr were collected 36 h post-transfection by centrifugation through 2 ml of a 20% sucrose cushion at 100,000g for 1 h at 4 °C in a SW-41 rotor (BeckmanCoulter). Cellular and released proteins were separated on a 12.5% SDS–PAGE and quantified by western blotting using an anti-Myc antibody and EPN-01 proteins purified from E. coli. The internal volume of each protein nanocage is around 3,000 nm3 (ref. 9), which is sufficient to package about 60 close-packed BlaM–Vpr molecules, assuming that each BlaM–Vpr molecule is approximately 50 nm3. Quantification of western blot band intensities indicated that an average of approximately 10 BlaM–Vpr molecules were actually packaged by each 60-subunit EPN-01* nanocage. For the BlaM delivery assay, 2 × 105 HeLa cells per well were seeded in 24-well plates. 24 h later, the indicated EPN quantities were added to cultures and incubated for 2 h at 37 °C. EPN-containing supernatants were replaced by CCF2-AM labelling media, prepared according to the manufacturer’s instructions (ThermoFisher) using CO -independent media (ThermoFisher) as the loading solution. Cells were labelled for 16 h at 13 °C and assayed by flow cytometry (FACSCanto, BD Biosciences) for changes in fluorescence emission spectrum from green (520 nm) to blue (447 nm). Data were collected with FACSDiva and analysed with FlowJo software (Treestar). Non-transduced cells treated with CCF2 were used to set the gate for uncleaved CCF2, which was set to discriminate transduced and non-transduced cells at a tolerance of <0.2% false positives (Extended Data Fig. 7d). Transduction assays were repeated independently at least three times (independent transfections). Standard deviations shown in Fig. 3 were calculated from three technical repeats of the BlaM delivery assay. Raw scans of all membranes and gels shown in the manuscript are included in this article as Supplementary Fig. 1. All other raw data are available from the corresponding authors upon request. Electron microscopy charge density maps, model fitting, and supporting data have been deposited in the EMDataBank under the accession number EMD-8278 (PDB accession number 5KP9). All novel plasmid constructs (Supplementary Information, Supplementary Table 3) have been submitted and are accessible through the Addgene plasmid repository ( https://www.addgene.org/).

News Article | February 15, 2017
Site: www.prweb.com

Worcester Polytechnic Institute will hold its second annual Advanced Biomanufacturing Symposium, a two-day, in-depth event that will focus on the technology and processes of continuous biomanufacturing and the challenges of making novel cell and regenerative tissue therapies that are approaching the clinic. The symposium, which was over-subscribed last year, is set for March 27–28, 2017. Organized by WPI life sciences and bioengineering faculty members and the university’s Biomanufacturing Education and Training Center (BETC), the symposium will bring together industry professionals and academic researchers working with new technologies, processes, and business practices that will have a significant impact on biomanufacturing in the near term. “2017 is shaping up to be an important year for biological products, with increasing public awareness of the industry and advances across the biomanufacturing spectrum that will demand our attention,” said Kamal Rashid, PhD, director of the BETC and research professor at WPI. “Evolving platforms and expression systems, progress towards end-to-end continuous biomanufacturing, the challenges of cell and tissue therapies—all of these topics will be explored in detail at our symposium.” This year’s keynote presenters include Manon Cox, PhD, president and chief executive officer of Protein Sciences Corp.; Jerome Ritz, MD, professor at Harvard Medical School and executive director of the Connell and O'Reilly Cell Manipulation and Gene Transfer Laboratory at Dana-Farber Cancer Institute; and Gail Naughton, PhD, chief executive officer of Histogen Inc. Of note, Kelvin Lee, PhD, Gore Professor of Chemical and Biomolecular Engineering at the University of Delaware, who led the team that organized the recently funded National Institute for Innovation in Manufacturing Biopharmaceuticals(NIIMBL), will also speak at the symposium. WPI is a member of NIIMBL. The symposium will feature session talks by subject matter experts from Biogen, Eppendorf, GE Healthcare, MilliporeSigma, Organovo, Pall Life Sciences, Sartorius Stedim Biotech, and Unum Therapeutics, as well as faculty members from Tufts University and WPI. “The talks will be presented in a single-track so participants will have access to all the content, and not have to choose between concurrent sessions,” Rashid said. “This worked very well last year. It helps maximize interaction and information exchange.” (Click here for photos from last year’s symposium. ) The symposium will take place in the Rubin Campus Center on WPI’s campus in Worcester, Mass. Registration is required and space is limited. (Click here for more event information and registration.) Funded in part by a grant from the Massachusetts Life Sciences Center, the BETC is a multi-faceted resource for the biologics industry, providing a range of hands-on customized programs. The BETC works with biomanufacturers to help them train, and retrain, their employees at a state-of-the-art center removed from their own production facilities. The center also provides research collaboration opportunities and consulting services to help companies manage challenges, explore new technologies, or scale up new processes. Founded in 1865 in Worcester, Mass., WPI is one of the nation’s first engineering and technology universities. Its 14 academic departments offer more than 50 undergraduate and graduate degree programs in science, engineering, technology, business, the social sciences, and the humanities and arts, leading to bachelor’s, master’s and doctoral degrees. WPI’s talented faculty work with students on interdisciplinary research that seeks solutions to important and socially relevant problems in fields as diverse as the life sciences and bioengineering, energy, information security, materials processing, and robotics. Students also have the opportunity to make a difference to communities and organizations around the world through the university’s innovative Global Projects Program. There are more than 45 WPI project centers throughout the Americas, Africa, Asia-Pacific, and Europe.

News Article | November 2, 2016
Site: www.nature.com

For recombinant protein overexpression in Escherichia coli, the clpP, mcsA, mcsB, clpC and clpC1–150 (NTD) genes/fragments from B. subtilis and clpA1–150 (NTD) from E. coli were cloned into pET21 or pET SUMO vectors (Novagen) conferring a terminal hexahistidine (6His) tag. Previously published pET21-derived plasmids were used for the expression of Geobacillus stearothermophilus McsB, YwlE and YwlED118N proteins13, 24, 25. For protein expression in B. subtilis, genes were cloned into the vector pHCMC05 (ref. 35) that contains the Psac IPTG-inducible promoter. For the expression of the ClpP, ClpPTRAP(S98A), ClpPX(E119A/R142E), ClpPX-TRAP(S98A/E119A/R142E), ClpC and ClpCEA(E32A/E106A), a C-terminal 6His tag including a Leu-Glu linker was introduced by PCR amplification. Single point mutations were generated using the QuikChange II mutagenesis kit (Agilent Technologies). The pHCMC05 plasmids described above were transformed into wild-type B. subtilis (strain 168) (ATCC 2385). To generate ClpC-knockout (∆clpC) bacteria, genomic DNA from a B. subtilis ∆clpC::tet strain36 was transformed into the B. subtilis strains containing the pHCMC05, pHCMC05-clpPX, pHCMC05-clpPX-TRAP, pHCMC05-ClpC or pHCMC05-ClpC-E32A-E106A plasmids. The disruption of clpC in the resulting strains was confirmed by sequencing. A previously published B. subtilis ∆clpP::specR (ref. 37) strain was used to grow ClpP-knockout bacteria for phosphoproteomic analysis. All bacterial cultures were grown in Luria–Bertani (LB) medium. For the B. subtilis ΔclpC and ΔclpP strains, tetracycline (10 μg ml−1) and spectinomycin (100 μg ml−1) were added, respectively. E. coli and B. subtilis cultures containing the pHCMC05-derived plasmids were cultured in the presence of ampicilin (50 μg ml−1) and chloramphenicol (10 μg ml−1), respectively. E. coli BL21 (DE3) containing pET21- or pET SUMO-derived vectors were cultured in the presence of ampicilin (50 μg ml−1). For the ClpPX-TRAP pull-down experiment in the wild-type background of B. subtilis (Fig. 1b), 6 independent B. subtilis cultures were grown in LB media expressing either ClpPX (3 control cultures to identify unspecific binding partners) or ClpPX-TRAP (3 sample cultures). After the cells were grown at 37 °C until mid-exponential phase, expression of His-tagged ClpPX or ClpPX-TRAP proteins was induced with 1 mM IPTG. Recombinant protein expression proceeded for 3 h at 37 °C. To induce the activity of heat-shock proteins, including McsB and various Clp ATPases, the cultures were incubated in a pre-warmed incubator at 45 °C for 45 min. Cells were collected by centrifugation, resuspended in lysis buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 10% glycerol) and stored at −80 °C. For the pull-down experiment comparing ClpP-trapped proteins in wild-type and ΔclpC backgrounds (Fig. 1c), the same procedure was applied using 12 independent B. subtilis cultures (3 wild type with ClpPX, 3 wild type with ClpPX-TRAP, 3 ΔclpC with ClpPX, 3 ΔclpC with ClpPX-TRAP). The thawed cell suspensions were incubated for 1 h on ice with 2 mg ml−1 lysozyme (Sigma), Complete protease inhibitor cocktail (Roche), 0.2 mM PMSF (Sigma) and 10 μg ml−1 DNase (Sigma). Cells were sonicated and the resultant lysate was cleared by centrifugation at 4 °C. For the purification of 6His-tagged ClpP, the lysate was incubated with Dynabeads His-Tag Isolation & Pulldown (Invitrogen) for 1 h at 4 °C. The beads were then washed 5× with lysis buffer and 2× with lysis buffer containing 50 mM imidazole. Two aliquots (5 or 10%) of the resulting beads were collected for SDS–PAGE analysis (protein elution with denaturing SDS–PAGE sample buffer) or Tris-acetate native-PAGE (protein elution with lysis buffer containing 500 mM imidazole). The remaining beads were subjected to reduction with 2 mM DTT (56 °C, 40 min), alkylation with 10 mM iodoacetamide (room temperature, in the dark, 45 min), and digestion with 2.5 μg Trypsin Gold (Promega) at 37 °C for 12 h. To analyse the mass of B. subtilis proteins co-purifying with ClpP(6His) under heat-shock conditions (Extended Data Fig. 1), matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI–TOF-MS) was performed. The corresponding protein purifications were spotted on a MALDI plate using a sinapinic acid (10 mg ml−1) matrix prepared in 50% acetonitrile (ACN) and 0.1% trifluoroacetic acid (TFA). The samples were measured in a 4800 MALDI-TOF-TOF (AB Sciex) instrument operated in linear mode. Calibration was performed internally using cytochrome c as standard. For the phosphoproteomic analysis of total cell extracts of B. subtilis ΔclpC, cells were lysed by sonication in buffer 4% SDS, 100 mM Tris, pH 7.5, 100 mM DTT and further processed using a filter aided sample preparation (FASP)38 modified method, as described previously15, followed by trypsin digestion at 37 °C for 12–16 h. Protein aggregates were dissolved in the SDS buffer and processed in the same manner. Trypsin digestion completion was inspected by retention time and UV intensity (214 nm) distribution upon reverse-phase high-performance liquid chromatography (RP-HPLC) separation of a 0.1% aliquot of the resulting supernatants on a monolithic column (Ultimate Plus equipped with a PepSwift PS-DVB column, 5 cm × 200 μm, Dionex-Thermo-Fisher). For the ClpP in vivo pull-down assays, a small aliquot (0.5%) of the on-bead trypsin digests was collected for subsequent quantitative analyses of co-purified proteins. The biological replicates were then pooled and further processed for phosphorylation analysis. Before phosphopeptide enrichment, sample digests were purified from buffer reagents by RP-C18 solid phase extraction at neutral pH using Oasis HLB cartridges (Waters). A previously described TiO protocol15, optimized in accordance to the acid-labile nature of phosphoarginine, was used for phosphopeptide enrichment. Reverse-phase separation of all peptide mixtures was carried out on an Ultimate 3000 RSLC nano-flow chromatography system (Thermo Scientific), using 0.5% acetic acid (pH 4.5 with NH ) as loading solvent, to prevent phosphoarginine hydrolysis during removal of salts in the pre-column (PepMapAcclaim C18, 5 mm × 0.3 mm, 5 μm, Thermo Scientific). Peptide separation was achieved on a C18 separation column (PepMapAcclaim C18, 50 cm × 0.75 mm, 2 μm, Thermo Scientific) by applying a linear gradient from 2% to 35% solvent B (80% ACN, 0.08% formic acid) in 120 or 240 min (pull-down and total extract samples, respectively) at a flow rate of 230 nl min−1. Solvent A was 2% ACN, 0.1% formic acid. The separation was monitored by UV detection and the outlet of the detector was directly coupled to the nano-electrospray ionization source (Proxeon Biosystems) for MS analysis. For phosphorylation analysis, TiO elution samples were infused into the LTQ Orbitrap Velos Pro ETD mass spectrometer (Thermo Scientific) using PicoTip nanospray emitter tips (New Objective) at a voltage of 1.5 kV. Peptides were analysed in data-dependent fashion in positive ionization mode, applying two different fragmentation methods: collision-induced dissociation (CID) and electron-transfer dissociation (ETD). The survey scan was acquired at resolution 60,000 and the 6 most abundant signals with charge state equal or higher than 2+ and exceeding an intensity threshold of 1,500 counts were selected for peptide fragmentation analysis. For MS/MS experiments, precursor ions were isolated within a 2.1-Da window centred on the observed m/z. To prevent repeated fragmentation of highly abundant peptides, selected precursors were dynamically excluded for 30 s from MS/MS analysis. CID fragmentation was achieved at normalized collision energy (NCE) of 35% with additional activation of the neutral loss precursor at M-49, M-32.7 and M-98 amu in a standard multistage activation method. For ETD, peptides were incubated with fluoranthene anions allowing for charge-state-dependent incubation times (90 ms for 3+ charged peptides), and resulting peptide fragments were detected in the ion trap analyser. For the identification of co-purified proteins in the ClpP in vivo pull-down assays, slightly different instrument settings were used. The 12 most abundant signals with charge state equal to or higher than 2+ and exceeding an intensity threshold of 500 counts were selected for CID peptide fragmentation analysis, applying an isolation window of 2 Da. Multistage activation was disabled. Selected precursors were dynamically excluded for 60 s from MS/MS analysis. Each pull-down digest was analysed twice, to evaluate technical reproducibility. For the phosphorylation analysis of TiO -enrchiment samples, raw data were extracted by the Proteome Discoverer software suite (version, Thermo Scientific) and searched against a combined forward/reversed database of B. subtilis Uniprot Reference Proteome with common contaminants added (4,455 entries in total) using MASCOT (version 2.2.07, Matrix Science). Carbamidomethylation of cysteine was set as fixed modification. Phosphorylation of serine, threonine, tyrosine and arginine plus oxidation of methionine were selected as variable modifications. Since tryptic cleavage is impaired at phosphorylated arginine, a maximum of two missed cleavage sites was allowed, whereas fully tryptic cleavage of both termini was required. The peptide mass deviation was set to 5 p.p.m.; fragment ions were allowed to have a mass deviation of 0.8 Da. False discovery rates were assessed using the Percolator tool39 within the Proteome Discoverer package. The results were filtered for peptide rank 1 and high identification confidence, corresponding to a 1% false discovery rate. Low-scoring peptides (Mascot ion score ≤ 20) were manually verified. In the rare cases in which a peptide was mapped to more than one protein sequence, both protein hits are reported. For reliable phosphorylation site analysis, all phosphopeptide hits were automatically re-analysed by the phosphoRS software40 within the Proteome Discoverer software suite. All the phosphopeptides identified in the ClpP in vivo pull-down assays were manually inspected. For other samples, we considered a phosphorylation site to be localized when the reported phosphoRS probability was higher than 90%. When multiple peptide-spectrum matches (PSMs) were obtained for the same phosphopeptide, only the PSM presenting the best identification/localization score compromise is presented. The multiple redundant PSMs were ranked according to their phosphoRS probability score into three categories (90–94%, 94–97% and 97–100%); the PSM presenting the best Mascot score within the highest phosphoRS category achieved was reported. PSMs presenting wrong or inconclusive localizations were thus excluded from the final list of phosphopeptides. Multiply phosphorylated peptides were also excluded from the analysis, because they cannot be classified into ‘phosphorylation type’ categories. For quantitative analysis of ClpP-trapped proteins, MS data were analysed using the MaxQuant software environment41, version, and its built-in Andromeda search engine42, against the B. subtilis Uniprot database described above. Strict trypsin specificity with up to two missed cleavages was used. The minimum required peptide length was set to six amino acids. Carbamidomethylation of cysteine was set as a fixed modification and N-acetylation of proteins N termini (42.010565 Da) and oxidation of methionine were set as variable modifications. During the main search, parent masses were allowed an initial mass deviation of 4.5 p.p.m. and fragment ions were allowed a mass deviation of 0.5 Da. The mass accuracy of the precursor ions was improved by time-dependent recalibration algorithms of MaxQuant. The ‘match between runs’ option was enabled to match identifications across samples within a time window of 2 min of the aligned retention times. The second peptide identification option in Andromeda was enabled. PSM and protein identifications were filtered using a target–decoy approach at false discovery rate of 1% for PSMs and 5% for proteins. Relative, label-free quantification of proteins was done using the MaxLFQ algorithm43 integrated into MaxQuant using default parameters. Unique and razor peptides were considered for quantification. Statistical evaluation of the resulting protein quantifications was performed using R scripting. Proteins quantified in less than 50% of the samples were filtered out. Missing LFQ values were substituted by the lowest value observed in the corresponding sample. For each protein, the fold change of LFQ-averaged intensities (ratio ClpPTRAP/control) and the corresponding P value (Limma test; Linear Models for Microarray Data) were calculated. A protein was considered to be a ClpP substrate when it was found to be enriched in the TRAP pull-down assays by a factor of at least 2 and P < 0.05. The ‘protein groups’ output file from MaxQuant containing the statistical evaluation is available in Supplementary Table 1. The mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository44 (http://proteomecentral.proteomexchange.org). Representative spectra of the pArg peptides identified in the ClpP trapping mutant pull-downs are presented in Supplementary Fig. 1. For the overexpression of recombinant proteins in E. coli BL21 (DE3), LB cultures were grown at 37 °C until the exponential phase, when expression was induced with 0.5 mM IPTG. After expression, cells were collected by centrifugation, resuspended in buffer A and stored at −80 °C. As the B. subtilis ClpC protein was unstable when expressed in E. coli, the production of wild-type ClpC(6His) and ClpCEA(6His) was performed in B. subtilis containing the corresponding pHCMC05 plasmids, by induction with 1 mM IPTG for 3 h at 37 °C. The optimal expression strategies and purification buffers are summarized in Extended Data Table 3. Cell suspensions were incubated on ice for 30 min in the presence of 1 mg ml−1 lysozyme, 0.1 mM PMSF, 10 μg ml−1 DNase and sonicated. Lysates were cleared by centrifugation and loaded on a 5 ml Ni- or Co-NTA column (GE Healthcare LifeSciences) equilibrated in buffer A. Washes were performed using a step-wise imidazole gradient, typically starting with 25 mM. The His-tagged proteins were eluted with buffer A containing 250 mM imidazole and concentrated using Vivaspin devices (Sartorius Stedim Biotech). Constructs expressed as a SUMO-fusion (SUMO-ClpP and SUMO-MecA) were incubated with SUMO Protease (Thermo Fisher Scientific) to obtain tag-free versions of the proteins. All resulting proteins were further purified by gel filtration on a Superdex-75 or -200 column (GE Healthcare LifeSciences) equilibrated with buffer B. For the purification of McsA(6His), the full-length protein was separated from an abundant cleavage product by ion exchange on a MonoQ column (GE Healthcare LifeSciences) using a 0.1–1 M NaCl gradient in 50 mM Tris, pH 8.5, 1 mM TCEP. All proteins were aliquoted and stored at −80 °C until further use. For the purification of the B. subtilis ClpCNTD and NTDEA mutant, affinity, ion exchange, and size exclusion chromatography were carried out in a 0.5× PBS buffer (6 mM Na/K phosphate, pH 7.25, 1.35 mM KCl, 68.5 mM NaCl). After elution from Ni-NTA using the PBS buffer supplemented with 250 mM imidazole, the protein was passed through a ResourceQ column (GE Healthcare LifeSciences). This was followed by gel filtration on a Superdex 200 column (GE Healthcare LifeSciences). Varying amounts of the analysed components (individual proteins, pArgAA) were incubated in 200 μl of reaction buffer (10 mM Tris, pH 8.0, 50 mM NaCl, 5 mM MgCl and 25 mM imidazole). After 5 min, 50 μl of Ni-Sepharose was added to capture His-tagged proteins. Sepharose was washed three times with 200 μl reaction buffer and bound proteins were eluted with 50 μl of elution buffer (10 mM Tris, pH 8.0, 50 mM NaCl, 5 mM MgCl and 1 M imidazole). For visualization, input and elution, fractions were analysed by SDS–PAGE. For the analysis of the oligomeric state of purified ClpP samples, a Tris-acetate non-denaturing PAGE system was used. Gel composition corresponded to 7, 10 or 15% acrylamide, 0.24% bis-acrylamide, 200 mM Tris-acetate, pH 7, polymerized in the presence of 0.042% ammonium persulfate (APS) and 0.125% N,N,N′,N′-tetramethylethylenediamine (TEMED). The running buffer composition was 25 mM Tris-HCl, 192 mM glycine, pH 8.3. Protein separation was performed at 4 °C and 150 mV for 3–4 h, and proteins were visualized by Coomassie-based InstantBlue protein stain (Expedeon). The NativeMark unstained protein standard (Thermo Fisher Scientific) was used for the estimation of ClpP oligomeric state. To produce the arginine-phosphorylated substrate, β-casein from bovine milk (Sigma) was incubated at 10 μM (all protein concentrations, if not otherwise mentioned, are for a single protomer) with 2 μM McsB and McsA from B. subtilis in 25 mM Tris, pH 7.5, 50 mM NaCl, 20 mM MgCl , and 5 mM ATP at 30 °C for 2 h. The reaction mixture was concentrated with a Vivaspin device and applied to a Superdex-200 size exclusion column equilibrated with 25 mM Tris, pH 7.5, 50 mM NaCl. The fractions most strongly enriched in β-casein over McsB (Extended Data Fig. 5a) were then pooled, concentrated and stored at −80 °C. The presence of the pArg modification was confirmed by immunoblotting using a pArg-specific antibody as described below. To obtain caseinpArg of higher purity, a similar phosphorylation reaction was performed, except that 4 μM of the G. stearothermophilus McsB(6His) was used instead of the B. subtilis McsBA complex. The reaction mixture was afterwards applied to a Ni-NTA column (GE Healthcare LifeSciences) to reduce the amounts of the His-tagged McsB before the final gel filtration purification step (Extended Data Fig. 5c). To prepare β-casein phosphorylated to different degrees (Fig. 4e), a large-scale phosphorylation reaction was set-up with the B. subtilis McsBA at 30 °C. An aliquot (time 0) was collected before the addition of ATP. After adding ATP, aliquots were taken after 10, 30, 60 and 120 min. After adding 100 mM EDTA (to stop phosphorylation), each aliquot was concentrated by Vivaspin ultrafiltration and applied to a Superdex 200 size exclusion column. An additional sample, which was collected after 120 min incubation with McsBA, was treated with 1 μM YwlE arginine phosphatase for 2 h. The phosphatase was then inactivated by adding 2 mM pervanadate and the sample was concentrated and submitted to size exclusion chromatography. The different caseinpArg preparations were concentrated and stored at −80 °C. To quantify the increase of arginine phosphorylation over time, a western blot was performed using the pArg-specific antibody described previously24. The caseinpArg preparations (2 μg) were separated by SDS–PAGE, transferred to a nitrocellulose membrane and fixed using a 0.4% formaldehyde solution in PBS, pH 7.5, for 30 min as previously described45. After blocking, the pArg-specific primary antibody (2 μg ml−1, Morphosys AG) was incubated overnight at 4 °C and the secondary antibody (goat anti human IgG F(ab′)2:HRP, AbD Serotec) was used at a 1:7,000 dilution for 1.5 h at room temperature. The detection was performed using ECL Plus western blotting substrate (Pierce). The signals were quantified using the ImageJ software46 and normalized to the band intensities observed in a coomassie-stained SDS–PAGE gel replicate having 1.3 μg of each protein preparation. In vitro degradation assays containing 0.16 μM ClpC (hexamer), 0.16 μM ClpP (heptamer) and 5 μM β-casein substrate were performed in 25 mM Tris, pH 7.5, 150 mM NaCl, 20 mM MgCl and 5 mM ATP at 30°C. A different β-casein concentration (10 μM) was used, for better resolution, when comparing ClpCP and ClpCPEA in MecA-dependent degradation. Small molecule compounds, for example, phospho-l-arginine (pArgAA, Toronto Biochemicals), l-arginine (Sigma) or sodium phosphate, pH 7.5, were added at 1 mM. Time-point aliquots were mixed with denaturing SDS–PAGE sample buffer containing 100 mM EDTA to stop the reaction and analysed by SDS–PAGE. The resulting gels were stained with InstantBlue dye (Expedeon) and quantified using ImageJ46. Supplementary Fig. 2 shows full SDS–PAGE gels and corresponding quantifications of Figs 2, 3, 4, 5. The radioactive kinase assay was performed at 19 °C in 25 mM Tris, pH 7.5, 50 mM NaCl, 2% glycerol, and 20 mM MgCl . 15 μM β-casein substrate was incubated with 1 μM McsB and/or other proteins (1 μM McsA, 5 μM YwlE) for 0, 30 and 120 min. The reaction was started by adding ATP (spiked with [γ-32P]ATP from Perkin Elmer) to a final concentration of 10 mM and stopped with denaturing SDS–PAGE sample buffer. After resolving the samples by SDS–PAGE, phosphorylation was visualized using phosphoimager technology (GE Healthcare Life Sciences). ATPase activity was determined by a coupled enzymatic reaction47. 0.125–0.5 μM ClpC was incubated with 18.75 U ml−1 pyruvate kinase, 21.45 U ml−1 lactate dehydrogenase, 0.2–0.3 mM NADH, 7.5 mM phosphoenolpyruvate and 2 mM ATP in 20 mM HEPES, pH 7.5, 100 mM NaCl, and 5 mM MgCl . Further assay proteins (MecA, McsB, McsA and β-casein) were added at 4–6-fold excess over ClpC. The absorption at 340 nm (A ) was recorded for 60 min using a Synergy H1 Multi-Mode Reader. The molar ATPase activity (v) was calculated by the equation: v = ∆A /(path length × 6,220 × [ClpC] × M−1 × cm−1). All activity data represent a minimum of three independent experiments and the variability is highlighted as standard deviation. ITC measurements were performed using VP-ITC (Microcal). Ligands (pArgAA, l-arginine, phospho-l-tyrosine (pTyrAA, from Sigma), and sodium phosphate, pH 7.5, were prepared at 0.3–1.4 mM in 25 mM Tris, pH 8, 50 mM NaCl, 0.2 mM TCEP and titrated to a 20 μM ClpCDWB (full-length) or 140 μM ClpCNTD protein solution present in the same buffer. The same set-up was used for the analysis of ClpANTD. The following settings were used: 5 μl (first) and 10 μl (all subsequent injections) injection volume, 300 s spacing time between the injections, 300 r.p.m. stirring speed, 25 °C temperature and overflow mode. Control experiments were carried out to correct for dilution effects upon protein/ligand titration. Resulting data were analysed with the MicroCal ORIGIN software. Crystals of a ClpCNTD–pArgAA complex were obtained by the sitting-drop vapour diffusion method upon mixing 100 nl of reservoir with 200 nl of a ClpCNTD (2 mM) protein solution containing 2 mM pArgAA. The optimized crystallization solution contained 13.5% (w/w) polyethylene glycol 4000, 500 mM ammonium sulfate, and 100 mM sodium acetate at pH 5. Crystals formed overnight at 19 °C and were soaked/cryo-protected in 40% polyethylene glycol 400, 20 mM Tris pH 8, and 6 mM pArgAA before being flash-frozen. Diffraction data to 1.6 Å were collected at 100 K using a wavelength of 0.9763 Å at beamline P14, DESY, Hamburg and integrated with XDS48. Molecular replacement in Phaser49 using ClpCNTD from B. subtilis (PDB code 2Y1Q; ref. 29) as a search model yielded a high-confidence solution. Refinement in CNS50, automatic rebuilding in Phenix51, and manual rebuilding in Coot52, 53 were carried out, followed by placing of ligands in Coot54. N(omega)-phospho-l-arginine structure and constraints were obtained with the respective SMILES code using eLBOW55. Rounds of refinement in Phenix56 and rebuilding in Coot yielded the final model with good statistics and geometry (Extended Data Table 2 and the following Ramachandran statistics: 98% favoured, 2% allowed, 0% outliers, 0% rotamer outliers). The featured-enhanced map, which is based on a composite residual omit map, was used to show ligand density. Figures were produced in Pymol57. For the phosphoproteomic analysis of B. subtilis protein aggregates, a 3-l culture of B. subtilis was grown at 37 °C until late exponential phase and then heat-shocked (50 °C) for 45 min. Cells were collected by centrifugation, resuspended in 25 mM Tris, pH 7.5, 150 mM NaCl, 0.5% Triton X-100 and stored at −80 °C. A 30-ml cell suspension in 25 mM Tris, pH 7.5, 150 mM NaCl, 0.5% Triton X-100 was incubated on ice with 3 mg ml−1 lysozyme, Complete protease inhibitor cocktail (Roche), 20 μg ml−1 DNase, 0.2 mM PMSF and 2 mM vanadate for 30 min. After dilution to 100 ml, cells were gently lysed at 4 °C by French Press (Constant Cell Disruption Systems) at 1.7 kbar. Lysis efficiency, estimated by plating out serial dilutions, exceeded 99%. The lysates were centrifuged at 45,000g for 30 min. The resulting pellets, containing insoluble protein aggregates, were further washed with 20 ml lysis buffer containing 0.4 mg ml−1 lysozyme, 10 μg ml−1 DNase, 0.2 mM PMSF and 2 mM vanadate. After 40 min homogenization at 4 °C under gentle agitation, the pellets were re-centrifuged. The protein aggregates contained in the pellets were then solubilized in 7 ml 25 mM Tris, pH 7.5, 8 M urea by sonication. The samples were again centrifuged to separate the urea-solubilized protein aggregates from cell debris. The resulting supernatants were stored at −80 °C until MS sample processing. To test the role of ClpC during heat stress, the following B. subtilis strains were investigated: wild type + pHCMC05, ∆clpC::tet + pHCMC05, ∆clpC::tet + pHCMC05-ClpC and ∆clpC::tet + pHCMC05-ClpCEA. For all experiments, cultures were grown at 37 °C in LB media containing 10 μg ml−1 chloramphenicol and 0.2 mM IPTG. After reaching exponential phase, the cultures were transferred to a pre-warmed incubator at 53 °C for 0, 30, 60 or 120 min, respectively. After heat stress, the samples were diluted sequentially and transferred to LB plates. To compare the survival rate after heat stress, we determined the number of colony-forming units (CFU). All experiments were independently performed three times and the observed variability is highlighted as standard deviation. Native mass spectrometry experiments were carried out on a Synapt G2Si instrument (Waters) with a nano-electrospray ionization (nESI) source. Mass calibration was performed by a separate infusion of NaI cluster ions. Solutions were ionised through a positive potential applied to metal-coated borosilicate capillaries (Thermo Scientific). β-casein samples (5 μM) were sprayed from 25 mM ammonium acetate, pH 6.8. The temperature settings were capillary voltage 1.5 kV, sample cone voltage 30 V, extractor source offset 46 V, and source temperature 50 °C. Data were processed using Masslynx V4.1 software. Source Data for Figs 1, 2, 3, 4, 5 are provided in the Supplementary Information. No statistical methods were used to predetermine sample size. The experiments were not randomized, and investigators were not blinded to allocation during experiments and outcome assessment.

DUBLIN--(BUSINESS WIRE)--Research and Markets has announced the addition of the "Top 10 Bioprocess Technology Market by Cell Culture, Cell Expansion, Cell Counting, Cell Line Development, Flow Cytometry, Single-Use Bioprocessing, Biologics Safety Testing, Tangential Flow Filtration, Virus Filtration & Region - Forecast to 2021" report to their offering. The top 10 bioprocess technology market is expected to USD 71.03 Billion by 2021 from USD 39.30 Billion in 2016, at a CAGR of 12.4% between 2016 and 2021. Growth in the biopharmaceutical industry, increase in R&D spending, rising demand for vaccine production, and technological advancements form important growth drivers for this market during the forecast period. The rising opportunities in emerging market and increasing pharmaceutical outsourcing provide significant growth opportunities for players operating in the top 10 bioprocess technology market. The report provides an overall understanding of the global top 10 bioprocess technology market. It segments the global market on the basis of type and region. On the basis of type, the market is segmented into cell culture, cell expansion, cell counting, cell line development, flow cytometry, single-use bioprocessing, biologics safety testing, virus filtration, tangential flow filtration, and pyrogen testing. In 2016, the cell culture segment is expected to account for the largest share of the global top 10 bioprocess technology market. The largest share of this segment is primarily attributed to the repeated purchase of consumables as compared to equipment and increase in funding for cell-based research. The major players in top 10 bioprocess technology market include GE Healthcare (U.S.), Danaher Corporation (U.S.), Thermo Fisher Scientific, Inc. (U.S.), Becton, Dickinson and Company (U.S.), Lonza Group AG (Switzerland), Merck Millipore (Germany), Sartorius Stedim Biotech S.A (France), Corning, Inc. (U.S.), Bio-Rad Laboratories (U.S.), and Charles River Laboratories (U.S.). For more information about this report visit http://www.researchandmarkets.com/research/jglv3h/top_10_bioprocess

News Article | February 23, 2017
Site: www.chromatographytechniques.com

Sartorius Stedim Biotech’s SARTOFLOW Smart is an easy benchtop crossflow system for optimized ultra- and dia-filtration applications. It can be used in many downstream processes, such as purification of vaccines, monoclonal antibodies and recombinant proteins. The system is suitable for flexible use in laboratory environments for process development and clinical trials, as well as for cGMP environments. It is equipped with a low shear 4-piston membrane pump that enables high product yields. In addition, the pump provides a wide range of flow rates, allowing users to choose between membrane surface areas from 50 cm² to as much as 0.14 m². The crossflow system is supplied with the company’s intuitive and easy to use DCU-4 control unit, which, when combined with the BioPAT SCADA MFCS-4 software, provides data logging and export. Its touchscreen offers instant access to all critical process parameters and displays control and alarm functions. A logbook function stores alarms, set points and user logs. Sartorius Stedim Biotech www.sartorius.com, 800-635-2906

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