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

1. Dyrk de tobaksplanter <ol><li> Skyl hvert mineraluld blok med 1 til 2 l deioniseret vand og derefter med 1 liter 0,1% [w / v] gødning opløsning. Placer en tobak frø i hver mineraluld blok og forsigtigt skylles med 0,25 liter gødning opløsning uden vask væk frøet 16. </li><li> Dyrk de tobaksplanter i 7 uger i et drivhus med 70% relativ fugtighed, en 16 timer fotoperiode (180 pmol sek - 1 m - 2 λ = 400-700 nm) og en 25/22 ° C lys / mørke temperatur regi...

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De tre metoder til varme nedbør beskrevet ovenfor effektivt kan fjerne tobak HCPS før enhver kromatografisk oprensning trin 16,17. De supplerer andre strategier, der tager sigte på at øge oprindelige produkt renhed, f.eks guttation 29, rhizosecretion 30 eller centrifugal udvinding 31,32, som alle er begrænset til udskilte proteiner. Imidlertid kan varme-baserede metoder kun anvendes på en meningsfuld måde, hvis målproteinet der skal oprenses kan modstå den mi...

Moderne sundhedssystemer i stigende afhængige af biofarmaceutiske proteiner 1. Producerer disse proteiner i planter er fordelagtig på grund af den lave patogen byrde og større skalerbarhed i forhold til konventionelle ekspressionssystemer 2-4. downstream (DSP) af planteafledte lægemidler kan imidlertid være udfordrende, fordi de forstyrrende ekstraktionsmetoder resulterer i en høj partikel byrde, med turbiditeter overstiger 5.000 nefelometrisk turbiditetsenheder (NTU&#39;er), og værtscelleprotein (HCP) koncentrationer ofte overstiger 95 % [m / m] 5,6. Udarbejde procedurer for præcisering er forpligtet til at fjerne spredte partikler 7-9, men kromatografi udstyr er billigere at operere i binde-og-elueringsmetode under indledende opsving produkt, hvis der er en tidligere trin til effektiv fjernelse af HCPS 10,11. Dette kan opnås enten ved udfældning målproteinet under anvendelse flocculmyrer 12 eller lav pH 13,14, samt ved at hjælpe HCPS at aggregere. Den selektive aggregering af ribulose-1,5-bisphosphat-carboxylase / oxygenase (RuBisCO), den mest udbredte HCP i grønne planter såsom tobak (Nicotiana tabacum), kan fremmes ved tilsætning af polyethylenglycol 15, men dette er dyrt og uforenelig med stor -skala produktion. Varmebehandling har vist sig at denaturere og bundfald mere end 95% af tobaksvarer HCPS, mens protein malaria vaccinekandidater såsom Vax8 forblive stabil i opløsning 16-18. Tre forskellige fremgangsmåder blev anvendt til at opnå den varme-igangsat bundfældning af tobaksvarer HCPS: (i) blanchering, dvs. nedsænkning af intakte blade i varm væske, (ii) en temperaturreguleret omrørt beholder, og (iii) en varmeveksler ( Figur 1) 16. For intakte blade, blanchering opnås en hurtig og effektiv udfældning af HCPS og var også letat skalere op og forenelige med eksisterende store fremstillingsprocesser, der omfatter et første skridt til at vaske plantebiomassen 19. I modsætning hertil temperaturstyrede skibe er allerede tilgængelige i nogle processer, og kan bruges til termisk behandling af planteekstrakter 20, men deres skalerbarhed og energi overførselshastighed er begrænset, fordi overfladen-til-volumen-forhold af tankene nedsættes gradvis og bliver uegnet til proces skala. En varmeveksler er en teknisk veldefineret alternativ til opvarmet omrørte fartøjer, men kræver en rigelig forsyning af opvarmning og afkøling medier, fx damp og koldt vand, samt et tæt kontrolleret volumetriske strømningshastighed, der er indrettet til varmeveksleren geometri og medier egenskaber, f.eks., den specifikke varmekapacitet. Denne artikel viser, hvordan alle tre metoder kan anvendes til varmeinduceret udfældning af tobak HCPS og planteprodukter HCPS generelt. Etablering og drift af EACh metode i et laboratorium indstilling kan anvendes til at vurdere deres egnethed til større målestok processer. Den største udfordring er at identificere passende skala-down modeller og driftsforhold for hver operation, der ligner enheder og betingelser, der anvendes under processen skala produktion. De data, der præsenteres her refererer til eksperimenter udført med transgene tobaksplanter, der udtrykker malariavaccine kandidat Vax8 og fluorescerende protein DsRed 16, men metoden har også været anvendt med succes til N. benthamiana planter transient udtrykker andre biofarmaceutiske proteiner 21. Et design-for-eksperimenter (DOE) nærmer 22 kan lette procesudvikling, og flokkuleringsmidler 23 kan også være en fordel i denne sammenhæng som tidligere beskrevet 8. Den væsentligste forskel mellem blanchering, opvarmede beholdere og varmevekslere er, at blanchering anvendes til intakte blade tidligt i processen hvorimod othendes anvendes på planteekstrakter (figur 1). <img alt="figur 1" src="/files/ftp_upload/54343/54343fig1.jpg" /> Figur 1:. Process Flow skema, der illustrerer gennemførelse af tre forskellige metoder til Tobacco HCP Heat Nedbør Plantematerialet vaskes og homogeniseres før afklaring og rensning. Udstyret til blancheringstrinnet (rød) kan let tilføjes til eksisterende maskineri. I modsætning hertil bruger en omrørt beholder (orange) og især en varmeveksler (blå) kræver en eller flere andre enheder og slanger. <a href="https://www.jove.com/files/ftp_upload/54343/54343fig1large.jpg" target="_blank">Klik her for at se en større version af dette tal.</a>

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			<td height="21" style="height:21px;width:360px;">2100P Portable Turbidimeter</td>
			<td style="width:195px;">Hach</td>
			<td style="width:119px;">4650000</td>
			<td style="width:352px;">Turbidimeter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Amine Coupling Kit</td>
			<td style="width:195px;">GE Healthcare</td>
			<td style="width:119px;">BR100050&#160;</td>
			<td>SPR chip coupling kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Autoclaving basket</td>
			<td style="width:195px;">Nalgene</td>
			<td style="width:119px;">6917-0230</td>
			<td>Basket for leaf blanching</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Biacore T200</td>
			<td style="width:195px;">GE Healthcare</td>
			<td style="width:119px;">28-9750-01</td>
			<td>SPR device</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Bio Cell Analyser BCA 003 R&#38;D with 3D ORM</td>
			<td style="width:195px;">Sequip</td>
			<td style="width:119px;">n.a.</td>
			<td>Particle size analyzer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Blender</td>
			<td style="width:195px;">Waring</td>
			<td style="width:119px;">800EG</td>
			<td>Blender</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">BP-410</td>
			<td style="width:195px;">Furh</td>
			<td style="width:119px;">2632410001</td>
			<td>Bag filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Centrifuge 5415D</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:119px;">5424 000.410</td>
			<td>Centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Centrifuge tube 15 mL</td>
			<td style="width:195px;">Labomedic</td>
			<td style="width:119px;">2017106</td>
			<td>Reaction tube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Centrifuge tube 50 mL self-standing</td>
			<td style="width:195px;">Labomedic</td>
			<td style="width:119px;">1110504</td>
			<td>Reaction tube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">CM5 chip</td>
			<td style="width:195px;">GE Healthcare</td>
			<td style="width:119px;">BR100012&#160;</td>
			<td>Chip for SPR measurements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Cuvette 10x10x45</td>
			<td style="width:195px;">Sarsted</td>
			<td style="width:119px;">67.754</td>
			<td>Cuvette for Zetasizer Nano ZS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Design-Expert(R) 8</td>
			<td style="width:195px;">Stat-Ease, Inc.</td>
			<td style="width:119px;">n.a.</td>
			<td>DoE software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Disodium phosphate</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">&#160;4984.3&#160;</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Ferty 2 Mega</td>
			<td style="width:195px;">Kammlott</td>
			<td style="width:119px;">5.220072</td>
			<td>Fertilizer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Forma -86C ULT freezer</td>
			<td style="width:195px;">ThermoFisher</td>
			<td style="width:119px;">88400</td>
			<td>Freezer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Greenhouse</td>
			<td style="width:195px;">n.a.</td>
			<td style="width:119px;">n.a.</td>
			<td>For plant cultivation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Grodan Rockwool Cubes 10x10cm</td>
			<td style="width:195px;">Grodan</td>
			<td style="width:119px;">102446</td>
			<td>Rockwool block</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Twentey-loop heat exchanger (4.8 m length)</td>
			<td style="width:195px;">n.a. (custom design)</td>
			<td style="width:119px;">n.a.</td>
			<td>Heat exchanger</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">HEPES</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">9105.3</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">K200P 60D</td>
			<td style="width:195px;">Pall</td>
			<td style="width:119px;">5302303</td>
			<td>Depth filter layer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">KS50P 60D</td>
			<td style="width:195px;">Pall</td>
			<td style="width:119px;">B12486</td>
			<td>Depth filter layer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Lauda E300</td>
			<td style="width:195px;">Lauda Dr Wobser GmbH</td>
			<td style="width:119px;">Z90010</td>
			<td>Water bath thermostat</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">L/S 24</td>
			<td style="width:195px;">Masterflex</td>
			<td style="width:119px;">SN-06508-24</td>
			<td>Tubing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:360px;">mAb 5.2</td>
			<td style="width:195px;">American Type Culture Collection</td>
			<td style="width:119px;">HB-9148</td>
			<td>Vax8 specific antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Masterflex L/S</td>
			<td style="width:195px;">Masterflex</td>
			<td style="width:119px;">HV-77921-75</td>
			<td>Peristaltic pump</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Miracloth</td>
			<td style="width:195px;">Labomedic</td>
			<td style="width:119px;">475855-1R</td>
			<td>Filter cloth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">MultiLine Multi 3410 IDS</td>
			<td style="width:195px;">WTW</td>
			<td style="width:119px;">WTW_2020</td>
			<td>pH meter / conductivity meter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Osram cool white 36 W</td>
			<td style="width:195px;">Osram</td>
			<td style="width:119px;">4930440</td>
			<td>Light source</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Phytotron</td>
			<td style="width:195px;">Ilka Zell</td>
			<td style="width:119px;">n.a.</td>
			<td>For plant cultivation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Sodium disulfit</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">8554.1</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Sodium chloride</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">P029.2</td>
			<td>Media component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:360px;">Stainless-steel vessel; 0.7-kg 2.0-L; height 180 mm; diameter 120 mm</td>
			<td style="width:195px;">n.a. (custom design)</td>
			<td style="width:119px;">n.a.</td>
			<td>Container for heat precipitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Synergy HT</td>
			<td style="width:195px;">BioTek</td>
			<td style="width:119px;">SIAFRT</td>
			<td>Fluorescence and spectrometric plate reader</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">VelaPad 60</td>
			<td style="width:195px;">Pall</td>
			<td style="width:119px;">VP60G03KNH4</td>
			<td>Filter housing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Zetasizer Nano ZS</td>
			<td style="width:195px;">Malvern</td>
			<td style="width:119px;">ZEN3600</td>
			<td>DLS particle size distribution measurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Zetasizer Software v7.11</td>
			<td style="width:195px;">Malvern</td>
			<td style="width:119px;">n.a.</td>
			<td>Software to operate the Zetasizer Nano ZS device</td>
		</tr>
	</tbody>
</table>

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

Planter ikke kun give fødevarer, foder og råvarer til mennesker, men er også blevet udviklet som et økonomisk system til biofarmaceutiske proteiner, såsom antistoffer, vaccinekandidater og enzymer produktion. Disse skal oprenses fra plantebiomassen men kromatografitrin hindres af de høje koncentrationer af værtscelleproteiner (HCPS) i planteekstrakter. Men de fleste HCPS irreversibelt aggregerer ved temperaturer over 60 ° C letter efterfølgende oprensning af målproteinet. Her er tre metoder præsenteres for at opnå den varme udfældning af tobak HCPS i enten intakte blade eller ekstrakter. Den blegning af intakte blade kan nemt indarbejdes i eksisterende processer, men kan have en negativ indvirkning på de efterfølgende filtrering trin. Det modsatte er tilfældet for varme udfældning af blade ekstrakter i en omrørt beholder, der kan forbedre ydeevnen af ​​nedstrøms operationer omend med større ændringer i procesudstyr design, såsomhomogenisator geometri. Endelig er en varmeveksler setup godt karakteriseret i form af varmeoverførsel betingelser og let at skala, men rengøring kan være svært, og der kan være en negativ indvirkning på filter kapacitet. Udformningen-of-eksperimenter tilgang kan bruges til at identificere de mest relevante procesparametre påvirker HCP fjernelse og produkt opsving. Dette letter anvendelsen af ​​hver metode i andre ekspressionssystemer platforme og identifikation af den mest egnede metode til en given oprensning strategi.

Heat udfældning af tobak værtscelleproteiner ved blanchering Fremgangsmåden beskrevet i afsnit 2. blanchering fremgangsmåde blev med held anvendt til udfældning HCPS fra tobaksblade med 70 ° C, hvilket reducerer TSP ved 96 ± 1% (n = 3), mens overvinde op til 51% af den Vax8 målproteinet, hvilket øger dens renhed fra 0,1% til 1,2% før kromatografisk separation 16. Det var også muligt at genvinde 83 ± 1% (n = 3) af det fluorescerende protein DsRed, ø...

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

Tre varme fældningsmetoder præsenteres som effektivt fjerner mere end 90% af værtscelleproteiner (HCPS) fra tobaksekstrakter forud for enhver anden oprensningstrin. Anlægget HCPS irreversibelt aggregerer ved temperaturer over 60 ° C.

Sammenligning af Tobak Host Cell Protein Removal Metoder ved Blanchering Intakte Planter eller ved varmebehandling af ekstrakter

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

Research

JoVE Journal

Biology

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 ...

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 ...

The MultiBac Protein Complex Production Platform at the EMBL

Den MultiBac Protein Complex Production Platform på EMBL

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

Protein-protein interaktioner visualiseret ved bimolekylær Fluorescens komplementering i tobaksprotoplaster og blade

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

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

In Vivo 4-Dimensional sporing af hæmatopoietisk stamcelletransplantation og stamcelle i Voksen Mouse calvarie knoglemarv

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

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Fækal Glukokortikoid Analyse: Non-invasive Adrenal Overvågning i dyr fra hestefamilien

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

DamID-seq: Genome-wide Kortlægning af protein-DNA interaktioner af High Throughput sekventering af adenin-methylerede DNA fragmenter

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

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts

Procedure for at vurdere effektiviteten af ​​flokkuleringsmidler til fjernelse af dispergerede partikler fra planteekstrakter

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used ...

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

Sammenligning af tre forskellige metoder til bestemmelse celleproliferation i brystcancercellelinier

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

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

Xenopus oocytter: Optimeret Metoder til mikroinjektion, Fjernelse af Follikulære cellelag og hurtig løsning Ændringer i Elektrofysiologiske Eksperimenter

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery ...