We would like to acknowledge Dr. Thomas Rademacher, Alexander Boes and Veronique Bei&#223; for providing the transgenic tobacco seeds, and Ibrahim Al Amedi for cultivating the tobacco plants. The authors wish to thank Dr. Richard M. Twyman for editorial assistance as well as G&#252;ven Edg&#252; for providing the MSP1-19 reference. This work was funded in part by the European Research Council Advanced Grant ''Future-Pharma'', proposal number 269110, the Fraunhofer-Zukunftsstiftung (Fraunhofer Future Foundation) and Fraunhofer-Gesellschaft Internal Programs under Grant No. Attract 125-600164.

<ol>
	<li>PhRMA. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 2013 Report: Medicines in Development - Biologics. , (2013).</li><li>Buyel, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Process+development+strategies+in+plant+molecular+farming.">Process development strategies in plant molecular farming.</a> Curr. Pharm. Biotechnol. 16, 966-982 (2015).</li><li>Stoger, E., Fischer, R., Moloney, M., Ma, J. K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+molecular+pharming+for+the+treatment+of+chronic+and+infectious+diseases.">Plant molecular pharming for the treatment of chronic and infectious diseases.</a> Annu. Rev. Plant Biol. 65, 743-768 (2014).</li><li>Melnik, S., Stoger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+factories+for+biopharmaceuticals.">Green factories for biopharmaceuticals.</a> Curr. Med. Chem. 20, 1038-1046 (2013).</li><li>Buyel, J. F., Twyman, R. M., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+and+downstream+processing+of+plant-derived+recombinant+proteins.">Extraction and downstream processing of plant-derived recombinant proteins.</a> Biotechnol. Adv. 33, 902-913 (2015).</li><li>Wilken, L. R., Nikolov, Z. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+and+purification+of+plant-made+recombinant+proteins.">Recovery and purification of plant-made recombinant proteins.</a> Biotechnol. Adv. 30, 419-433 (2012).</li><li>Buyel, J. F., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scale-down+models+to+optimize+a+filter+train+for+the+downstream+purification+of+recombinant+pharmaceutical+proteins+produced+in+tobacco+leaves.">Scale-down models to optimize a filter train for the downstream purification of recombinant pharmaceutical proteins produced in tobacco leaves.</a> Biotechnol. J. 9, 415-425 (2014).</li><li>Buyel, J. F., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flocculation+increases+the+efficacy+of+depth+filtration+during+the+downstream+processing+of+recombinant+pharmaceutical+proteins+produced+in+tobacco.">Flocculation increases the efficacy of depth filtration during the downstream processing of recombinant pharmaceutical proteins produced in tobacco.</a> Plant Biotechnol. J. 12, 240-252 (2014).</li><li>Buyel, J. F., Opdensteinen, P., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellulose-based+filter+aids+increase+the+capacity+of+depth+filters+during+the+downstream+processing+of+plant-derived+biopharmaceutical+proteins.">Cellulose-based filter aids increase the capacity of depth filters during the downstream processing of plant-derived biopharmaceutical proteins.</a> Biotechnol. J. 10, 584-591 (2014).</li><li>Buyel, J. F., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generic+chromatography-based+purification+strategies+accelerate+the+development+of+downstream+processes+for+biopharmaceutical+proteins+produced+in+plants.">Generic chromatography-based purification strategies accelerate the development of downstream processes for biopharmaceutical proteins produced in plants.</a> Biotechnol. J. 9, 566-577 (2014).</li><li>Buyel, J. F., Woo, J. A., Cramer, S. M., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+quantitative+structure-activity+relationship+models+to+develop+optimized+processes+for+the+removal+of+tobacco+host+cell+proteins+during+biopharmaceutical+production.">The use of quantitative structure-activity relationship models to develop optimized processes for the removal of tobacco host cell proteins during biopharmaceutical production.</a> J. Chromatogr. A. 1322, 18-28 (2013).</li><li>Holler, C., Vaughan, D., Zhang, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyethyleneimine+precipitation+versus+anion+exchange+chromatography+in+fractionating+recombinant+beta-glucuronidase+from+transgenic+tobacco+extract.">Polyethyleneimine precipitation versus anion exchange chromatography in fractionating recombinant beta-glucuronidase from transgenic tobacco extract.</a> J. Chromatogr. A. 1142, 98-105 (2007).</li><li>Buyel, J. F., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Downstream+processing+of+biopharmaceutical+proteins+produced+in+plants:+the+pros+and+cons+of+flocculants.">Downstream processing of biopharmaceutical proteins produced in plants: the pros and cons of flocculants.</a> Bioengineered. 5, 138-142 (2014).</li><li>Hassan, S., van Dolleweerd, C. J., Ioakeimidis, F., Keshavarz-Moore, E., Ma, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Considerations+for+extraction+of+monoclonal+antibodies+targeted+to+different+subcellular+compartments+in+transgenic+tobacco+plants.">Considerations for extraction of monoclonal antibodies targeted to different subcellular compartments in transgenic tobacco plants.</a> Plant Biotechnol. J. 6, 733-748 (2008).</li><li>Arfi, Z. A., Drossard, J., Hellwig, S., Fischer, R., Buyel, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyclonal+antibodies+can+effectively+detect+tobacco+host+cell+proteins+after+RuBisCO+depletion+and+endotoxin+removal.">Polyclonal antibodies can effectively detect tobacco host cell proteins after RuBisCO depletion and endotoxin removal.</a> Biotechnol. J. , (2015).</li><li>Buyel, J. F., Gruchow, H. M., Boes, A., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+of+a+host+cell+protein+heat+precipitation+step+simplifies+the+subsequent+purification+of+recombinant+proteins+from+tobacco.">Rational design of a host cell protein heat precipitation step simplifies the subsequent purification of recombinant proteins from tobacco.</a> Biochem. Eng. J. 88, 162-170 (2014).</li><li>Buyel, J. F., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+juice+extractor+can+simplify+the+downstream+processing+of+plant-derived+biopharmaceutical+proteins+compared+to+blade-based+homogenizers.">A juice extractor can simplify the downstream processing of plant-derived biopharmaceutical proteins compared to blade-based homogenizers.</a> Process Biochem. 50, 859-866 (2014).</li><li>Beiss, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat-precipitation+allows+the+efficient+purification+of+a+functional+plant-derived+malaria+transmission-blocking+vaccine+candidate+fusion+protein.">Heat-precipitation allows the efficient purification of a functional plant-derived malaria transmission-blocking vaccine candidate fusion protein.</a> Biotechnol. Bioeng. 112, 1297-1305 (2015).</li><li>Ma, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+approval+and+a+first-in-human+phase+I+clinical+trial+of+a+monoclonal+antibody+produced+in+transgenic+tobacco+plants.">Regulatory approval and a first-in-human phase I clinical trial of a monoclonal antibody produced in transgenic tobacco plants.</a> Plant Biotechnol. J. 13, 1106-1120 (2015).</li><li>Mahajan, P. V., Caleb, O. J., Singh, Z., Watkins, C. B., Geyer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postharvest+treatments+of+fresh+produce.">Postharvest treatments of fresh produce.</a> Philos T R Soc A. 372, (2014).</li><li>Menzel, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+blanching+reduces+the+host+cell+protein+content+and+substantially+enhances+the+recovery+and+stability+of+two+plant+derived+malaria+vaccine+candidates.">Optimized blanching reduces the host cell protein content and substantially enhances the recovery and stability of two plant derived malaria vaccine candidates.</a> Front. Plant Sci. , (2015).</li><li>Buyel, J. F., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+complex+systems+using+the+design+of+experiments+approach:+transient+protein+expression+in+tobacco+as+a+case+study.">Characterization of complex systems using the design of experiments approach: transient protein expression in tobacco as a case study.</a> J. Vis. Exp. , e51216 (2014).</li><li>Buyel, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Procedure+to+evaluate+the+efficiency+of+flocculants+for+the+removal+of+dispersed+particles+from+plant+extracts.">Procedure to evaluate the efficiency of flocculants for the removal of dispersed particles from plant extracts.</a> J. Vis. Exp. , e53940 (2016).</li><li>Simonian, M. H., Smith, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrophotometric+and+colorimetric+determination+of+protein+concentration.">Spectrophotometric and colorimetric determination of protein concentration.</a> Curr. Protoc. Mol. Biol. 76, (2006).</li><li>Buyel, J. F., Kaever, T., Buyel, J. J., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predictive+models+for+the+accumulation+of+a+fluorescent+marker+protein+in+tobacco+leaves+according+to+the+promoter/5'UTR+combination.">Predictive models for the accumulation of a fluorescent marker protein in tobacco leaves according to the promoter/5'UTR combination.</a> Biotechnol. Bioeng. 110, 471-482 (2013).</li><li>Piliarik, M., Vaisocherova, H., Homola, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+plasmon+resonance+biosensing.">Surface plasmon resonance biosensing.</a> Methods Mol. Biol. 503, 65-88 (2009).</li><li>Kim, T. D., Ryu, H. J., Cho, H. I., Yang, C. H., Kim, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+behavior+of+proteins:+Heat-resistant+proteins+and+their+heat-induced+secondary+structural+changes.">Thermal behavior of proteins: Heat-resistant proteins and their heat-induced secondary structural changes.</a> Biochemistry-Us. 39, 14839-14846 (2000).</li><li>Kwon, S., Jung, Y., Lim, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+heat-stable+proteins+in+Escherichia+coli.">Proteomic analysis of heat-stable proteins in Escherichia coli.</a> Bmb Rep. 41, 108-111 (2008).</li><li>Komarnytsky, S., Borisjuk, N. V., Borisjuk, L. G., Alam, M. Z., Raskin, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+recombinant+proteins+in+tobacco+guttation+fluid.">Production of recombinant proteins in tobacco guttation fluid.</a> Plant Physiol. 124, 927-933 (2000).</li><li>Drake, P. M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+rhizosecretion+as+a+production+system+for+recombinant+proteins+from+hydroponic+cultivated+tobacco.">Development of rhizosecretion as a production system for recombinant proteins from hydroponic cultivated tobacco.</a> FASEB J. 23, 3581-3589 (2009).</li><li>Turpen, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+mosaic+virus+and+the+virescence+of+biotechnology.">Tobacco mosaic virus and the virescence of biotechnology.</a> Philos. Trans. R. Soc. Lond., Ser. B: Biol. Sci. 354, 665-673 (1999).</li><li>Kingsbury, N. J., McDonald, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Evaluation+of+E1+Endoglucanase+Recovery+from+Tobacco+Leaves+Using+the+Vacuum+Infiltration-Centrifugation+Method.">Quantitative Evaluation of E1 Endoglucanase Recovery from Tobacco Leaves Using the Vacuum Infiltration-Centrifugation Method.</a> Biomed. Res. Int. , (2014).</li><li>Buyel, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Numeric+simulation+can+be+used+to+predict+heat+transfer+during+the+blanching+of+leaves+and+intact.">Numeric simulation can be used to predict heat transfer during the blanching of leaves and intact.</a> Biochem. Eng. J. , (2015).</li><li>Mandal, M. K., Fischer, R., Schillberg, S., Schiermeyer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+protease+activity+by+antisense+RNA+improves+recombinant+protein+production+in+Nicotiana+tabacum+cv.+Bright+Yellow+2+(BY-2)+suspension+cells.">Inhibition of protease activity by antisense RNA improves recombinant protein production in Nicotiana tabacum cv. Bright Yellow 2 (BY-2) suspension cells.</a> Biotechnol. J. 9, 1065-1073 (2014).</li><li>Welty, J. R., Wicks, C. E., Wilson, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fundamentals of momentum, heat, and mass transfer. , (1976).</li><li>Lowe, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aggregation,+stability,+and+formulation+of+human+antibody+therapeutics.">Aggregation, stability, and formulation of human antibody therapeutics.</a> Advances in protein chemistry and structural biology. 84, 41-61 (2011).</li><li>Gong, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+human+antibody+constant+domains+with+increased+stability.">Engineered human antibody constant domains with increased stability.</a> J. Biol. Chem. 284, 14203-14210 (2009).</li><li>Rouet, R., Lowe, D., Christ, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+engineering+of+the+human+antibody+repertoire.">Stability engineering of the human antibody repertoire.</a> FEBS Lett. 588, 269-277 (2014).</li><li>Stabel, J. R., Lambertz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+pasteurization+conditions+for+the+inactivation+of+Mycobacterium+avium+subsp+paratuberculosis+in+milk.">Efficacy of pasteurization conditions for the inactivation of Mycobacterium avium subsp paratuberculosis in milk.</a> J. Food Prot. 67, 2719-2726 (2004).</li><li>Wichers, H., Mills, C., Wichers, H., Hoffmann-Sommergruber, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+12.">Ch. 12.</a> Managing Allergens in Food. , 336 (2006).</li><li>Davis, P. J., Williams, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+modification+by+thermal+processing.">Protein modification by thermal processing.</a> Allergy. 53, 102-105 (1998).</li><li>Dubois, M. F., Hovanessian, A. G., Bensaude, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat-shock-induced+denaturation+of+proteins.+Characterization+of+the+insolubilization+of+the+interferon-induced+p68+kinase.">Heat-shock-induced denaturation of proteins. Characterization of the insolubilization of the interferon-induced p68 kinase.</a> J. Biol. Chem. 266, 9707-9711 (1991).</li></ol>

著者らは、開示する利害の競合がありません。

上記の熱沈殿のための3つの方法が効果的に任意のクロマトグラフィー精製ステップ16,17の前にタバコのHCPを削除することができます。彼らは、分泌タンパク質に限定されているすべてのそれらの初期生成物の純度を高めることを目指して他の戦略、 例えば、排水29、rhizosecretion 30または遠心抽出31,32を 、補完します。標的タンパク質は、以上1分〜60°Cの...

現代の医療システムは、ますますバイオ医薬品タンパク質1に依存しています。植物におけるこれらのタンパク質を産生することは、従来の発現系2-4と比較して、低い病原体負荷とスケーラビリティに有利で ​​す。破壊的な抽出手順は5,000比濁分析濁度単位（NTUs）を超える濁度、及び宿主細胞タンパク質（HCP）濃度は、多くの場合、95を超えると、高い粒子負荷をもたらすしかし、植物由来の医薬品の下流の処理（DSP）は、挑戦することができ％[メートル/メートル] 5,6。 精巧な浄化手順は、分散粒子7-9を削除する必要があります。ただし、クロマトグラフィー装置はのHCP 10,11を効率的に除去するための以前のステップがある場合、最初の製品の回収時にバインドおよび溶出モードで動作するように安価であるされています。これはflocculを使用して標的タンパク質を沈殿させることによってのいずれかで達成することができますアリ12または低pH 13,14、ならびにのHCPが凝集させることもできます。リブロース-1,5-二リン酸カルボキシラーゼ/オキシゲナーゼ（のRuBisCO）の選択的な凝集は、例えば、タバコ（ タバコ ）などの緑色植物の中で最も豊富なHCPは、ポリエチレングリコール15を追加することによって促進することができるが、これは高価であり、大規模なと互換性がありません-scale製造。例えばVax8などのタンパク質のマラリアワクチン候補は、溶液中で安定16-18まま熱処理は、タバコのHCPの95％以上を変性し、沈殿させることが示されています。 （I）ブランチング、 すなわち、高温の液体中の無傷の葉の浸漬、（ii）の温度制御撹拌容器、および（iii）熱交換器（：三つの異なるアプローチがタバコのHCPの熱誘導沈殿を達成するために使用しました。 図1）16。無傷の葉のために、ブランチングは、のHCPの迅速かつ効率的な降水量を達成し、また簡単でしたスケールアップと植物バイオマス19を洗浄するための最初のステップを含む、既存の大規模製造プロセスに適合します。対照的に、温度制御された血管は、既にいくつかのプロセスで利用可能であり、タンクの表面対体積比が徐々に低下するため、プラントの熱処理20を抽出し 、それらのスケーラビリティとエネルギー移動速度が制限されるために使用することができるとプロセスのスケールで適さなくなります。熱交換器は、撹拌容器を加熱するために技術的に明確に定義された代替手段であるが豊富な加熱の供給およびメディア、 例えば、蒸気、冷水、冷却、ならびに熱交換器の形状に適合されている厳密に制御体積流量を必要としメディアの特性、 例えば 、特定の熱容量。この記事では、すべての3つの方法が一般的にタバコのHCP、および植物のHCPの熱による沈殿のために使用することができる方法を示しています。 EACの設立と運営実験室の設定における時間法は、大規模なプロセスのためのそれらの適合性を評価するために使用することができます。主要な課題は、プロセス規模製造中に使用されるデバイスおよび条件に似ている各操作に対して十分な縮小モデルと運転条件を識別することです。ここに提示したデータは、マラリアワクチン候補Vax8を発現するトランスジェニックタバコ植物を用いて行った実験を参照し、蛍光タンパク質は、16のDsRedが、方法はまた、成功裏にNに適用されていますbenthamiana植物は一過他のバイオ医薬品タンパク質21を発現しています 。 デザイン・オブ・実験（DOE）アプローチ22は、プロセスの開発を容易にすることができ、以前8に記載のように凝集剤23もまた、この状況において有益であり得ます。ブランチング、加熱された容器と熱交換器との間の主な違いは、ブランチングは、初期のOT一方過程で無傷の葉に適用されることです彼女は、植物抽出物（ 図1）に適用されます。 <img alt="図1" src="/files/ftp_upload/54343/54343fig1.jpg" /> 図1： タバコHCP熱降水のための3つの異なる方法の実施例を示すプロセスフロースキームは、植物材料を洗浄し、明確化し、精製前の均質化されています。ブランチング工程（赤色）のための装置は、容易に既存の機械に追加することができます。これとは対照的に、攪拌容器（橙）、特に熱交換器（青）を使用して、1つまたはいくつかの追加の機器や配管を必要とします。 <a href="https://www.jove.com/files/ftp_upload/54343/54343fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

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</table>

comparison of tobacco host cell protein removal methods by blanching intact plants or by heat treatment of extracts

植物は人間のための食品、飼料、原材料を提供するだけでなく、このような抗体、ワクチン候補と酵素などのバイオ医薬品タンパク質、のための経済的な生産システムとして開発されているだけではなく。これらは、植物バイオマスから精製されなければならないが、クロマトグラフィー工程は、植物抽出物中の宿主細胞タンパク質（のHCP）の高濃度によって妨げられます。しかし、ほとんどののHCPは、不可逆的に、標的タンパク質のその後の精製を容易にする60℃以上の温度で凝集します。ここでは、3つの方法は、無傷の葉または抽出物のいずれかでタバコのHCPの熱沈殿を達成するために提示されます。無傷の葉のブランチングを容易に既存のプロセスに組み込むことができるが、その後の濾過工程に悪影響を与えることができます。反対は、次のようなプロセス装置の設計に大きな変化、とはいえ下流操作のパフォーマンスを向上させることができ、撹拌容器、中葉抽出物の熱沈殿のために真でありますホモジナイザージオメトリ。最後に、熱交換器の設定は、熱伝達条件および拡張が容易という点で十分に特徴付けられているが、洗浄が困難であることができ、フィルタの能力に負の影響があってもよいです。デザイン・オブ・実験アプローチは、HCPの除去および生成物回収に影響を与える最も関連するプロセスパラメータを識別するために使用することができます。これは、他の発現プラットフォーム内の​​各メソッドと与えられた精製戦略に最も適した方法の特定のアプリケーションを容易にします。

ブランチングによってタバコ宿主細胞タンパク質の熱沈殿 項2に記載のブランチング手順が成功したタバコは96±1％TSPを減らす、70°Cでの葉からのHCPを沈殿させるために使用した（N = 3）従って、Vax8標的タンパク質の51％まで回復し、そのを増加させながらクロマトグラフィー分離16前の0.1％から1.2％まで純度。それ64.1％と3.3％の純度を増加させる?...

Watch this Scientific Journal Video about Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts at JoVE.com

Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts

三熱沈殿法は、効果的に前に他の精製工程にタバコ抽出物から、宿主細胞タンパク質（のHCP）の90％以上を除去することが示されています。植物のHCPは、不可逆的に60℃以上の温度で凝集します。

無傷の植物をブランチングすることによって、または抽出物の熱処理によりタバコ宿主細胞タンパク質の除去方法の比較

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical ...

comparison-tobacco-host-cell-protein-removal-methods-blanching-intact

Research

JoVE Journal

Biology

9.0K ビュー.  Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V.. 三熱沈殿法は、効果的に前に他の精製工程にタバコ抽出物から、宿主細胞タンパク質（のHCP）の90％以上を除去することが示されています。植物のHCPは、不可逆的に60℃以上の温度で凝集します。 

ビデオ: Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

免疫手術でヒト胚性幹細胞の導出

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

生物学

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

アフリカツメガエル卵抽出物の調製と分画

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

ELISAによるネイティブ無傷ヌクレオソーム上の翻訳後修飾の検出

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

EMBLでMultiBacタンパク質複合体生産プラットフォーム

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

タバコプロトプラストと葉で二分子蛍光相補性によって可視化し、タンパク質間相互作用

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

成体マウス頭蓋冠骨髄中の造血幹細胞および前駆細胞のin vivo 4次元トラッキングで

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Generation of Transgenic Hydra by Embryo Microinjection

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology1. Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.

Generation of Transgenic Hydra by Embryo Microinjection

Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi.

トランスジェニックの世代<em&gt;ハイドラ</em&gt;胚マイクロインジェクションによる

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糞便グルココルチコイド分析：ウマにおける非侵襲性副腎モニタリング

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.

We describe herein an assay by coupling DNA adenine methyltransferase identification (DamID) to high throughput sequencing (DamID-seq). This improved method provides a higher resolution and a wider dynamic range, and allows analyzing DamID-seq data in conjunction with other high throughput sequencing data such as ChIP-seq, RNA-seq, etc.

DamID-配列：アデニンメチル化DNA断片のハイスループットシークエンシングによるタンパク質-DNA相互作用のゲノムワイドなマッピング

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is ...

Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility with high-throughput formatting. This protocol describes the use of three different methods for measuring cell proliferation in vitro including conventional hemocytometer counting chamber, a luminescence-based assay that utilizes the change in the metabolic activity of viable cells as a measure of the relative number of cells, and a multi-mode cell imager that measures cell number using a counting algorithm. Each method presents its own advantages and disadvantages for the measurement of cell proliferation, including time, cost and high-throughput compatibility. This protocol demonstrates that each method could accurately measure cell proliferation over time, and was sensitive to detect growth at differing cellular densities. Additionally, measurement of cell proliferation using a cell imager was able to provide further information such as morphology, confluence and allowed for a continual monitoring of cell proliferation over time. In conclusion, each method is capable of measuring cell proliferation, but the chosen method is user-dependent.

This protocol describes the use of three different methods for analyzing cell proliferation in breast cancer cell lines. This includes the use of conventional cell counting, luminescence-based cell viability, and cell counting through the use of a cell imager. Each offers advantages for the reproducible measurement of cell proliferation.

乳癌細胞株における細胞増殖を決定するための3つの異なる方法の比較

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility ...