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. dyrke tobakksplanter <ol><li> Spyle hver mineralullblokk med 1 til 2 l avionisert vann og deretter med 1 liter 0,1% [w / v] gjødsel-løsning. Plasser en tobakk frø i hver mineralull blokk og forsiktig flush med 0,25 liter gjødsel løsning uten å vaske bort frø 16. </li><li> Dyrke tobakksplanter i 7 uker i et veksthus med 70% relativ fuktighet, en 16 timers fotoperiode (180 umol sek - 1 m - 2; λ = 400-700 nm) og en 25/22 ° C lys / mørke temperaturregime....

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De tre metoder for varme nedbør beskrevet ovenfor effektivt kan fjerne tobakk Helsepersonell før noen kromatografisk rensetrinn 16,17. De utfyller andre strategier som tar sikte på å øke første produktet renhet, f.eks guttation 29, rhizosecretion 30 eller sentrifugal utvinning 31,32, som alle er begrenset til utskilte proteiner. Imidlertid kan de varmebaserte metoder bare brukes på en meningsfull måte dersom målet proteinet som skal renses kan tåle den minst...

Moderne helsevesen stadig avhenge biofarmasøytisk proteiner 1. Fremstilling av disse proteiner i planter er fordelaktig på grunn av den lave patogen byrde og større skalerbarhet forhold til konvensjonelle ekspresjonssystemer 2-4. Imidlertid kan nedstrømsprosessering (DSP) av plante-avledet legemidler være utfordrende fordi forstyrrende ekstraksjonsmetoder resultere i en høy partikkel belastning, med turbidities stiger 5000 nefelometriske turbiditetsenheter (NTUs), og vertscelleprotein (HCP) konsentrasjoner ofte overstiger 95 % [m / m] 5,6. Forseggjort avklarings prosedyrer er nødvendig for å fjerne dispergerte partikler 7-9, men kromatografi utstyr er mindre kostbart å operere i binde-og-elute modus under innledende produktgjenvinningen hvis det er et tidligere trinn for effektiv fjerning av HCPs 10,11. Dette kan oppnås enten ved utfelling av målproteinet ved hjelp flocculmaur 12 eller lav pH 13,14, samt ved forårsaker Helsepersonell å aggregere. Den selektive aggregering av ribulose-1,5-bisfosfat-karboksylase / oksygenase (RUBISCO), det mest tallrike HCP i grønne planter som tobakk (Nicotiana tabacum) kan fremmes ved tilsetning av polyetylenglykol 15, men dette er kostbart og uforenlig med stor -skala produksjon. Varmebehandling har vist seg å denaturere og presipitere mer enn 95% av tobakks HCPs, mens protein malaria vaksinekandidater som Vax8 forbli stabil i oppløsning 16-18. Tre forskjellige metoder ble brukt for å oppnå den varmeinduserte utfelling av tobakks HCPs: (i) blanchering, dvs. nedsenking av intakte blader i varm væske, (ii) en temperaturkontrollert omrørt beholder, og (iii) en varmeveksler ( Figur 1) 16. For intakte blader, forvelling oppnås rask og effektiv utfelling av Helsepersonell og var også lettå skalere opp og kompatibel med eksisterende storskala produksjonsprosesser som inkluderer et første skritt for å vaske anlegget biomasse 19. I motsetning til dette temperaturstyrt fartøy er allerede tilgjengelig i noen prosesser og kan anvendes for varmebehandling av plante-ekstrakter 20, men deres skalerbarhet og energioverføring hastighet er begrenset, fordi den overflate-til-volum-forholdet av tankene er gradvis redusert og blir uegnet i prosessen skala. En varmeveksler er en teknisk veldefinert alternativ til å varmes opp omrørt fartøy, men krever en rikelig tilførsel av varme- og kjølemedier, for eksempel damp og kaldt vann, samt en kontrollert volumetrisk strømningshastighet som er tilpasset til varmeveksleren geometri og media egenskaper, f.eks., den spesifikke varmekapasitet. Denne artikkelen viser hvordan alle tre fremgangsmåter kan anvendes for varmeinduserte utfelling av tobakk HCPs, og plante HCPs generelt. Etablering og drift av EACh fremgangsmåte i et laboratorium kan brukes til å vurdere om de er egnet for større skala prosesser. Den store utfordringen er å identifisere tilstrekkelig skala-down modeller og driftsforhold for hver operasjon som ligner enhetene og betingelsene som brukes under prosessen-skala produksjon. Dataene presentert her viser til eksperimenter utført med transgene tobakksplanter som uttrykker det malaria vaksine kandidat Vax8 og fluorescerende protein DsRed 16, men fremgangsmåten er også blitt brukt til N. benthamiana planter forbigående uttrykker andre biofarmasøytisk proteiner 21. En design-of-eksperimenter (DoE) nærmer 22 kan forenkle prosessutvikling, og flokulanter 23 kan også være nyttig i denne sammenheng som tidligere beskrevet åtte. Hovedforskjellen mellom blanche, oppvarmede beholdere og varmevekslere er at blanche påtrykkes intakte bladene tidlig i prosessen, mens othennes tilføres planteekstrakter (figur 1). <img alt="Figur 1" src="/files/ftp_upload/54343/54343fig1.jpg" /> Figur 1:. Process Flow skjema som illustrerer gjennomføring av tre ulike metoder for Tobacco HCP Heat Nedbør Plantematerialet blir vasket og homogenisert før avklaring og rensing. Utstyr for forvellingstrinn (rød) lett kan legges til eksisterende maskineri. I motsetning til ved hjelp av en rørt fartøy (oransje) og spesielt en varmeveksler (blå) krever en eller flere andre enheter og rør. <a href="https://www.jove.com/files/ftp_upload/54343/54343fig1large.jpg" target="_blank">Klikk her for å se en større versjon av dette tallet.</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 gir ikke bare mat, fôr og råvarer for mennesker, men har også blitt utviklet som et økonomisk produksjonssystem for biofarmasøytisk proteiner, slik som antistoffer, vaksinekandidater og enzymer. Disse må renses fra plantebiomasse men kromatografiske trinn blir hindret av de høye konsentrasjonene av vertscelleproteiner (HCPs) i planteekstrakter. Men de fleste HCPs irreversibelt å aggregere ved temperaturer over 60 ° C som letter etterfølgende rensing av målproteinet. Her er tre metoder presenteres for å oppnå varme utfelling av tobakk Helsepersonell i enten intakte blader eller ekstrakter. Blanche av intakte bladene kan enkelt bli innlemmet i eksisterende prosesser, men kan ha en negativ innvirkning på etterfølgende filtreringstrinn. Det motsatte er tilfelle for varme utfelling av blad ekstrakter i en rørt fartøy, som kan forbedre ytelsen til nedstrømsvirksomheten riktignok med store endringer i prosessutstyr design, for eksempelhomogenisator geometri. Til slutt blir en varmeveksler oppsett godt karakterisert med hensyn til varmeoverføringsbetingelser og lett å skala, men rengjøring kan være vanskelig og det kan være en negativ innvirkning på filterkapasitet. Utformingen-of-eksperimenter tilnærming kan brukes til å identifisere de mest relevante prosessparametre som påvirker HCP fjerning og produktgjenvinning. Dette letter anvendelsen av hver metode i andre uttrykk plattformer og identifisering av den mest egnede metode for et gitt rensestrategi.

Heat utfelling av tobakk vertscelleproteiner forvelling Blancheringsfremgangsmåten beskrevet i avsnitt 2 ble med hell anvendt for å utfelle HCPs fra tobakksblader med 70 ° C, noe som reduserer TSP ved 96 ± 1% (n = 3), mens utvinning av opptil 51% av den Vax8 målproteinet, og dermed øke dens renhet fra 0,1% til 1,2% før kromatografisk separasjon 16. Det var også mulig å utvinne 83 ± 1% (n = 3) av det fluorescerende protein DsRed, øker dens renhet fra ...

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 utfellingsmetoder er presentert som effektivt fjerner mer enn 90% av vertscelleproteiner (HCPs) fra tobakksekstrakter før en hvilken som helst annen rensetrinn. Anlegget HCPs irreversibelt å aggregere ved temperaturer over 60 ° C.

Sammenligning av Tobakks Host Cell Protein fjerning Metoder ved Blanche Intakte Planter eller ved varmebehandling av 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 produksjonsplattform 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 interaksjoner visualisert ved bimolekylær fluorescens complementation i Tobakk Protoplaster og blader

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 av Hematopoietiske Stem og stamceller i Adult Mouse Calvarial Bone Marrow

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

Fecal Glukokortikoid analyse: Ikke-invasiv Adrenal Monitoring i Equids

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 Kartlegging av Protein-DNA Interaksjoner med høy gjennomstrømning Sekvensering av adenin-metylert 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

Prosedyre for å vurdere effektiviteten av Fnokker for fjerning av dispergert 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 av tre forskjellige metoder for bestemmelse Cell Proliferation i brystkreftcellelinjer

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: Optimalisert Metoder for Mikroinjeksjon, Fjerning av Follikulære cellelag, og rask løsning Endringer i Elektro 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 ...