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3 pages, 1 figure, 1 table, 4 references. ; Recombinant gene strategies using fusion tags for purification are essential procedures to obtain large protein quantities. However, most cloning systems result in recombinant proteins with added amino acids inexistent in their native forms which can lead to significant changes in protein properties. An original and simple cloning strategy is proposed to obtain proteins identical in amino acid sequence to the native proteins. ; The authors acknowledge funding from the Government of Andalusia (Spain) BIO288. M. Santana was recipient of grant SFRH/BPD/34404/2006 from the Fundação para a Ciência e a Tecnologia (Portugal). ; Peer reviewed

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium's collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form. ; This work was supported by Goethe University (Corona funds), the DFG-funded CRC: "Molecular Principles of RNA-Based Regulation," DFG infrastructure funds (project numbers: 277478796, 277479031, 392682309, 452632086, 70653611), the state of Hesse (BMRZ), the Fondazione CR Firenze (CERM), and the IWB-EFRE-program 20007375. This project has received funding from the European Union's Horizon 2020 research and innovation program under Grant Agreement No. 871037. AS is supported by DFG Grant SCHL 2062/2-1 and by the JQYA at Goethe through project number 2019/AS01. Work in the lab of KV was supported by a CoRE grant from the University of New Hampshire. The FLI is a member of the Leibniz Association (WGL) and financially supported by the Federal Government of Germany and the State of Thuringia. Work in the lab of RM was supported by NIH (2R01EY021514) and NSF (DMR-2002837). BN-B was supported by theNSF GRFP.MCwas supported byNIH (R25 GM055246 MBRS IMSD), and MS-P was supported by the HHMI Gilliam Fellowship. Work in the labs of KJ and KT was supported by Latvian Council of Science Grant No. VPP-COVID 2020/1-0014. Work in the UPAT's lab was supported by the INSPIRED (MIS 5002550) project, which is implemented under the Action "Reinforcement of the Research and Innovation Infrastructure," funded by the Operational Program "Competitiveness, Entrepreneurship and Innovation" (NSRF 2014–2020) and cofinanced by Greece and the EU (European Regional Development Fund) and the FP7 REGPOT CT-2011- 285950–"SEE-DRUG" project (purchase of UPAT's 700MHz NMR equipment). Work in the CM-G lab was supported by the Helmholtz society. Work in the lab of ABö was supported by the CNRS, the French National Research Agency (ANR, NMRSCoV2- ORF8), the Fondation de la Recherche Médicale (FRM, NMR-SCoV2-ORF8), and the IR-RMN-THC Fr3050 CNRS. Work in the lab of BM was supported by the Swiss National Science Foundation (Grant number 200020_188711), the Günthard Stiftung für Physikalische Chemie, and the ETH Zurich. Work in the labs of ABö and BM was supported by a common grant from SNF (grant 31CA30_196256). This work was supported by the ETHZurich, the grant ETH40 18 1, and the grant Krebsliga KFS 4903 08 2019. Work in the lab of the IBS Grenoble was supported by the Agence Nationale de Recherche (France) RA-COVID SARS2NUCLEOPROTEIN and European Research Council Advanced Grant DynamicAssemblies. Work in the CA lab was supported by Patto per il Sud della Regione Siciliana–CheMISt grant (CUP G77B17000110001). Part of this work used the platforms of the Grenoble Instruct-ERIC center (ISBG; UMS 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-05-02) and GRAL, financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE- 0003). Work at the UW-Madison was supported by grant numbers NSF MCB2031269 and NIH/NIAID AI123498. MM is a Ramón y Cajal Fellow of the Spanish AEI-Ministry of Science and Innovation (RYC2019-026574-I), and a "La Caixa" Foundation (ID 100010434) Junior Leader Fellow (LCR/BQ/PR19/11700003). Funded by project COV20/00764 fromthe Carlos III Institute of Health and the SpanishMinistry of Science and Innovation to MMand DVL. VDJ was supported by the Boehringer Ingelheim Fonds. Part of this work used the resources of the Italian Center of Instruct-ERIC at the CERM/ CIRMMP infrastructure, supported by the Italian Ministry for University and Research (FOE funding). CF was supported by the Stiftung Polytechnische Gesellschaft. Work in the lab of JH was supported by NSF (RAPID 2030601) and NIH (R01GM123249). ; Peer reviewed

This work was supported by ERANET-IB08-007 project from the European Union and its linked national project EUI2008- 03610 to AV. We also appreciate the support from EME2007-08 to NFM from Universitat Autonoma de Barcelona, from Antartide 2010 to MLT and EP, from MIUR Azioni Integrate Italia-Spagna 2010 Prot. IT10LECLM9 to MLT, from MINECO (IT2009-0021) to AV and LT, from AGAUR (2009SGR-108) to AV. AV is also supported by The Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN, Spain), an initiative funded by the VI National R&D&i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. PS has received predoctoral fellowship from ISCIII, and AV has been distinguished with an ICREA ACADEMIA award (Catalonia, Spain). ; Obtaining high levels of pure proteins remains the main bottleneck of many scientific and biotechnological studies. Among all the available recombinant expression systems, Escherichia coli facilitates gene expression by its relative simplicity, inexpensive and fast cultivation, well-known genetics and the large number of tools available for its biotechnological application. However, recombinant expression in E. coli is not always a straightforward procedure and major obstacles are encountered when producing many eukaryotic proteins and especially membrane proteins, linked to missing posttranslational modifications, proteolysis and aggregation. In this context, many conventional and unconventional eukaryotic hosts are under exploration and development, but in some cases linked to complex culture media or processes. In this context, alternative bacterial systems able to overcome some of the limitations posed by E. coli keeping the simplicity of prokaryotic manipulation are currently emerging as convenient hosts for protein production. We have comparatively produced a "difficult-to-express" human protein, the lysosomal enzyme alpha-galactosidase A (hGLA) in E. coli and in the psychrophilic bacterium Pseudoalteromonas haloplanktis TAC125 cells (P. haloplanktis TAC125). While in E. coli the production of active hGLA was unreachable due to proteolytic instability and/or protein misfolding, the expression of hGLA gene in P. haloplanktis TAC125 allows obtaining active enzyme. These results are discussed in the context of emerging bacterial systems for protein production that represent appealing alternatives to the regular use of E. coli and also of more complex eukaryotic systems.

[Background] Rhipicephalus (Boophilus) spp. ticks economically impact on cattle production in Africa and other tropical and subtropical regions of the world. Tick vaccines constitute a cost-effective and environmentally friendly alternative to tick control. The R. microplus Bm86 protective antigen has been produced by recombinant DNA technology and shown to protect cattle against tick infestations. ; [Results] In this study, the genes for Bm86 (R. microplus), Ba86 (R. annulatus) and Bd86 (R. decoloratus) were cloned and characterized from African or Asian tick strains and the recombinant proteins were secreted and purified from P. pastoris. The secretion of recombinant Bm86 ortholog proteins in P. pastoris allowed for a simple purification process rendering a final product with high recovery (35–42%) and purity (80–85%) and likely to result in a more reproducible conformation closely resembling the native protein. Rabbit immunization experiments with recombinant proteins showed immune cross-reactivity between Bm86 ortholog proteins. ; [Conclusion] These experiments support the development and testing of vaccines containing recombinant Bm86, Ba86 and Bd86 secreted in P. pastoris for the control of tick infestations in Africa. ; This work was supported by the Wellcome Trust under the Animal Health in the Developing World initiative through project 0757990 entitled "Adapting recombinant anti-tick vaccines to livestock in Africa" and the Consejería de Educación y Ciencia, JCCM, Spain (project PAI06-0046-5285) and was facilitated through the Integrated Consortium on Ticks and Tick-borne Diseases (ICTTD-3), financed by the International Cooperation Program of the European Union, coordination action project No. 510561. V. Naranjo was funded by Junta de Comunidades de Castilla–La Mancha (JCCM), Spain. ; Peer reviewed

8 pages,2 figures and 2 tables ; [Background] The cattle ticks, Boophilus spp., affect cattle production in tropical and subtropical regions of the world. Tick vaccines constitute a cost-effective and environmentally friendly alternative to tick control. The recombinant B. microplus Bm86 protective antigen has been shown to protect cattle against tick infestations. Recently, the gene coding for B. annulatus Bm86 ortholog, Ba86, was cloned and the recombinant protein was secreted and purified from the yeast Pichia pastoris. ; [Results] Recombinant Ba86 (Israel strain) was used to immunize cattle to test its efficacy for the control of B. annulatus (Mercedes, Texas, USA strain) and B. microplus (Susceptible, Mexico strain) infestations. Bm86 (Gavac and Mozambique strain) and adjuvant/saline were used as positive and negative controls, respectively. Vaccination with Ba86 reduced tick infestations (71% and 40%), weight (8% and 15%), oviposition (22% and 5%) and egg fertility (25% and 50%) for B. annulatus and B. microplus, respectively. The efficacy of both Ba86 and Bm86 was higher for B. annulatus than for B. microplus. The efficacy of Ba86 was higher for B. annulatus (83.0%) than for B. microplus (71.5%). The efficacy of Bm86 (Gavac; 85.2%) but not Bm86 (Mozambique strain; 70.4%) was higher than that of Ba86 (71.5%) on B. microplus. However, the efficacy of Bm86 (both Gavac and Mozambique strain; 99.6%) was higher than that of Ba86 (83.0%) on B. annulatus. ; [Conclusion] These experiments showed the efficacy of recombinant Ba86 for the control of B. annulatus and B. microplus infestations in cattle and suggested that physiological differences between B. microplus and B. annulatus and those encoded in the sequence of Bm86 orthologs may be responsible for the differences in susceptibility of these tick species to Bm86 vaccines. ; This work was supported by the Wellcome Trust under the Animal Health in the Developing World initiative through project 0757990 entitled "Adapting recombinant anti-tick vaccines to livestock in Africa" and the Consejería de Educación y Ciencia, JCCM, Spain (project PAI06-0046-5285) and was facilitated through the Integrated Consortium on Ticks and Tick-borne Diseases (ICTTD-3), financed by the International Cooperation Program of the European Union, coordination action project No. 510561. ; Peer reviewed

We previously reported that the expression of Bombyx mori 30Kc19 gene in CHO cells significantly improved both the production and sialylation of recombinant human EPO (rHuEPO) in adhesion culture mode. In this study, the effects of 30Kc19 expression and supplementation of 30Kc19 recombinant protein on the productivity and glycosylation pattern of rHuEPO were investigated in the serum-free suspension culture mode. Especially, glycosylation pattern was examined in detail using a quantitative MALDI-TOF MS method. The expression of 30Kc19 increased the EPO production by 2.5-folds and the host cells produced rHuEPO with more complex glycan structures and a larger content of sialic acid and fucose. The glycan structures of rHuEPO in the 30Kc19-expressing cell consisted of bi-, tri-, tetra-, and penta-antennary branching (35, 18, 33, and 14 %, respectively), while the control cells produced predominantly bi-antennary branching (70 %). About 53 % of the glycans from rHuEPO in the 30Kc19-expressing cell was terminally sialylated, while no obvious sialylated glycan was found in the control cells. The percentage of fucosylated glycans from the 30Kc19-expressing cell culture was 77 %, whereas only 61 % of the glycans from the control cell were fucosylated glycans. We also examined whether these effects were observed when the recombinant 30Kc19 protein produced from Escherichia coli was supplemented into the culture medium for CHO cells. In the control cell line without the 30Kc19 gene, EPO production increased by 41.6 % after the addition of 0.2 mg/mL of the recombinant 30Kc19 protein to the culture medium. By the Western blot analysis after two-dimensional electrophoresis (2-DE) of isoforms of EPO, we confirmed that 30Kc19 enhanced the sialylation of EPO glycans. These results demonstrated that both 30Kc19 gene expression and the recombinant 30Kc19 protein addition enhanced rHuEPO productivity and glycosylation in suspension culture. In conclusion, the utilization of 30Kc19 in CHO cell culture holds great promise for use in the manufacturing of improved biopharmaceutical glycoproteins. ; This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (nos. 2011-0000331, 2011K000682, and 2011-0020984). This work was also supported by the Pioneer Research Program and by Basic Science Research Program through the NRF grant funded by the Ministry of Education, Science, and Technology (nos. 2011-0001643 and 2010-0002520). This work was also supported by WCU (World Class University) program through the Korea Science and Engineering Foundation funded by the Ministry of Education, Science and Technology (R32-2010-000-10213-0) and by a grant of the Korea Healthcare technology R&D Project, Ministry of Health & Welfare, Republic of Korea (grant no. A103017). ; OAIID:oai:osos.snu.ac.kr:snu2012-01/102/0000002410/14 ; SEQ:14 ; PERF_CD:SNU2012-01 ; EVAL_ITEM_CD:102 ; USER_ID:0000002410 ; ADJUST_YN:Y ; EMP_ID:A002014 ; DEPT_CD:458 ; CITE_RATE:3.425 ; FILENAME:(2012.11)Enhancement of recombinant human EPO production and.pdf ; DEPT_NM:화학생물공학부 ; EMAIL:thpark@snu.ac.kr ; SCOPUS_YN:Y ; CONFIRM:Y

Protein-based therapeutics are highly successful in clinic and currently enjoy unprecedented recognition of their potential. More than 100 genuine and similar number of modified therapeutic proteins are approved for clinical use in the European Union and the USA with 2010 sales of US$108 bln; monoclonal antibodies (mAbs) accounted for almost half (48%) of the sales. Based on their pharmacological activity, they can be divided into five groups: (a) replacing a protein that is deficient or abnormal; (b) augmenting an existing pathway; (c) providing a novel function or activity; (d) interfering with a molecule or organism; and (e) delivering other compounds or proteins, such as a radionuclide, cytotoxic drug, or effector proteins. Therapeutic proteins can also be grouped based on their molecular types that include antibody-based drugs, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytics. They can also be classified based on their molecular mechanism of activity as (a) binding non-covalently to target, e.g., mAbs; (b) affecting covalent bonds, e.g., enzymes; and (c) exerting activity without specific interactions, e.g., serum albumin. Most protein therapeutics currently on the market are recombinant and hundreds of them are in clinical trials for therapy of cancers, immune disorders, infections, and other diseases. New engineered proteins, including bispecific mAbs and multispecific fusion proteins, mAbs conjugated with small molecule drugs, and proteins with optimized pharmacokinetics, are currently under development. However, in the last several decades, there are no conceptually new methodological developments comparable, e.g., to genetic engineering leading to the development of recombinant therapeutic proteins. It appears that a paradigm change in methodologies and understanding of mechanisms is needed to overcome major challenges, including resistance to therapy, access to ...

One of the expectations of science, from both substantive and network perspectives, is its cumulative nature. It is a strong hope that scientific research and analysis promote an extension of knowledge as a function of critique and as a stimulus for extension. We are pleased that the comments of Curd, Duster, Lappé, and Mazur help us (and, we hope, others) better understand and appreciate our case study.

'Europa ist ein Laboratorium für die Rekombination unterschiedlicher Aspekte von citizenship. Der Text schlägt als analytischen Raster drei Dimensionen (Mitgliedschaft, Rechte und Praktiken) und drei Konzeptionen (liberale, republikanische und kommunitäre) von citizenship vor. Im zweiten Abschnitt werden drei Herausforderungen für eine monistische Auffassung von Staatsbürgerschaft als homogener Status und exklusive Bindung zwischen Individuum und einer einzigen politischen Gemeinschaft diskutiert. Internationale Migrationen schaffen überlappende multiple Bürgerschaften, die sich in doppelter Staatsangehörigkeit und 'Wohnbürgerrechten' niedergelassener AusländerInnen manifestieren. Die Forderungen nationaler Minderheiten nach territorialer Autonomie führen zu einer Föderalisierung zentralistischer Staaten, wodurch eine ineinander verschachtelte Mehrebenen-Bürgerschaft geschaffen wird, wie sie in anderer Weise auch in der Europäischen Union entsteht. Kulturelle Bürgerrechte werden entlang von Gruppenzugehörigkeiten differenziert, indem kulturellen Minderheiten Schutz vor Diskriminierung, besondere Ausnahmen von allgemeinen Bürgerpflichten oder öffentliche Ressourcen und Anerkennung zugestanden wird.' (Autorenreferat)

Explores the PROFETAS programme for development of a more sustainable food system by studying the feasibility of substituting meat with plant based alternatives. This book places emphasis on improving the food system by reducing the use of energy, land, and freshwater, at the same time limiting the impacts on health and animal welfare