Questo lavoro è sostenuto dall'Associazione per la promozione dell'innovazione giovanile CAS (2021084) e dal Fondo di sostegno al progetto di distribuzione chiave del piano d'azione triennale dell'Istituto di microbiologia, Accademia cinese delle scienze.

1. Preparazione delle piante di N. benthamiana
<ol><li>Mettere i blocchi di piantina in un vassoio, aggiungere 1 L di acqua e immergere per circa 2 ore fino a completa saturazione.</li>
	<li>Cospargere uniformemente i semi di N. benthamiana selvatici sui blocchi della piantina, coprire con un coperchio e germinare per 1 settimana. Coltiva N. benthamiana in serra con fotoperiodo di 18 ore, 24 °C, 45% di umidità relativa e concima ogni 2 settimane.</li>
	<li>Una volta che i semi sono germogliati, usa una pinzetta per rimuovere delicatamente una piantina, facendo attenzione a non danneggiare le sue radici. Scava una piccola fossa al centro di un nuovo vassoio, mettici dentro la piantina e copri leggermente le radici con la terra. Coprite con un coperchio e continuate a crescere.</li>
	<li>Cresci fino a quando le piantine hanno tre foglie, con il coperchio. Togliete il coperchio quando emerge la 4a fogliavera.</li>
	<li>Lasciare che le piante continuino a crescere per altre due settimane prima dell'agroinfiltrazione.</li>
</ol>2. Costruzione della GFP-His ricombinante
<ol><li>Eseguire l'ottimizzazione del codone di N. benthamiana e la sintesi di GFP e PR1a (MGFVLFSQLPSFLLVSTLLLFLVISHSCRAG) in base alle sequenze geniche.</li>
	<li>Effettuare la doppia digestione del vettore pCAMBIA1300 utilizzando gli enzimi Xba I e Sac I (vedi Tabella dei Materiali).</li>
	<li>Eseguire l'amplificazione di PR1a utilizzando primer a monte (TTGGAGAGAACACGGGGGACTCTAGAATGGGATT TGTTCTCTTTTC) e primer a valle (TCCTCG CCCTTGCTCACCATGTCGACCCCGGCACGGCAAGA GTGGG), amplificazione della GFP mediante primer a monte (CCCACTCTTGCCGTGCCGGGGGGTCGACATGGTGAG CAAGGGCGAGGA) e primer a valle (GGGGAAATTCGAGCTCAGTGGTGATGGTGGTG GTGAGATCTTCCGGACTTGT).</li>
	<li>Utilizzando la ricombinazione omologa, clonare PR1a e GFP nel vettore di espressione della pianta pCAMBIA1300. Dopo il trasferimento in E. coli, selezionare i singoli cloni e inviarli all'azienda per il sequenziamento. Il confronto tra i risultati del sequenziamento e il plasmide giusto è il plasmide ricombinante pCAMBIA1300-PR1a-GFP-His, indicato come p35s-PR1a-GFP-His.</li>
</ol>3. Produzione di GFP-His in N. benthamiana
<ol><li>Aggiungere 1 μL di plasmide GFP in 50 μL di cellule del recettore dell'Agrobacterium GV3101, mescolare delicatamente e aggiungere alla coppa dell'elettrodo, rimuovere dopo l'elettrocuzione sull'elettroshocker.</li>
	<li>Distribuire l'Agrobacterium tumefaciens trasformato su una piastra di terreno solido LB contenente 50 μg/mL di kanamicina (Kan) e 50 μg/mL di rifampicina (Rif). Incubare capovolto a 28 °C per 48 h. Il vettore pCAMBIA1300 è kan-resistente e l'Agrobacterium è resistente al rif; solo l'Agrobacterium corretto può crescere su piastre con kan e rif. I cloni monoclonali erano i trasformanti positivi, denominati A. tumefaciens GV3101-p35s-PR1a-GFP-His (AtGPG).</li>
	<li>Usa uno stuzzicadenti sterilizzato per prelevare una quantità appropriata di AtGPG e inocularela in 4 ml di LB liquido con Kan e Rif. Coltura a 28 °C, 220-300 giri/min per 24 h.</li>
	<li>Sottocoltura dei batteri 1:50 in LB fresco (50 μg/mL Kan, 10 mM 2-(N-Morpholino) acido etansolfonico (MES), 20 μM acetosiringone (AS)) e incubare a 28 °C, 220-300 rpm per 10-12 h.</li>
	<li>Raccogliere i batteri mediante centrifugazione a 1500 x g per 10 minuti e risospendere il pellet con 1 mL di MMA (10 mM MgCl2, 10 mM MES pH 5,6, 100 μM AS).</li>
	<li>Misura e regola la densità ottica (OD) di AtGPG a OD600=0,8 utilizzando MMA. Incubare i batteri per altre 2-4 ore a temperatura ambiente. Aspirare la soluzione batterica con una siringa senza ago, premerla leggermente contro la parte posteriore delle foglie e fare pressione per far penetrare la soluzione nelle foglie. Inietta solo le 3-4 foglie centrali per pianta.</li>
	<li>Continua a coltivare N. benthamiana in serra per 3-4 giorni prima di raccogliere le foglie per l'estrazione delle proteine.</li>
</ol>4. Osservazione della localizzazione subcellulare della GFP
<ol><li>Dopo 48 h di agroinfiltrazione, selezionare un'anta piana e utilizzare un punzone da 6 mm per bloccare la lama per la punzonatura.</li>
	<li>Versare la soluzione di saccarosio al 30% su un vetrino, posizionare il campione di foglia con la parte posteriore rivolta verso l'alto e coprirlo con un vetro di copertura.</li>
	<li>Impostare la lunghezza d'onda di eccitazione su 488 nm e rilevare la fluorescenza verde a 490-550 nm dopo un ingrandimento di 400x. Scatta la prima foto una volta preparata la diapositiva e un'altra 10 minuti dopo.</li>
</ol>5. Estrazione della GFP dalla pianta
<ol><li>Estrazione AWF<ol><li>Raccogliere le foglie fresche intatte di N. benthamiana che esprimono GFP ricombinante a 3 giorni dall'agroinfiltrazione. Trattare delicatamente la foglia e mantenerne l'integrità per evitare l'efflusso di proteine intracellulari. Usa immediatamente le foglie raccolte per il passaggio successivo per evitare la degradazione delle proteine.</li>
		<li>Sciacquare la lama della foglia con ddH2O sterilizzato preraffreddato per rimuovere i detriti.</li>
		<li>Pesare le foglie fresche e immergere 20 g di foglie (lato abassiale rivolto verso il basso) in 100 mL di tampone fosfato 100 mM pre-raffreddato (PB 100 mM Na2HPO4 e 100 mM NaH2PO4 sono stati configurati separatamente e miscelati a un rapporto di 2,2:1 pH 7,0) in un becher a bocca larga da 200 mL. Coprire le foglie con filato di nylon a maglia fine da 100 maglie (vedi Tabella dei materiali) e una piastra di plastica. NOTA: Il pre-raffreddamento impedisce la degradazione delle proteine. I volumi tampone possono essere regolati in base alla scala sperimentale. Assicurati che tutte le foglie siano immerse.</li>
		<li>Posizionare il becher in una pompa a vuoto. Applicare il vuoto a 0,8 MPa per 1 minuto, quindi ripristinare rapidamente la pressione atmosferica.</li>
		<li>Rimuovere le foglie dal tampone e asciugare delicatamente con carta assorbente. Arrotolare le foglie, avvolgerle con il filo a rete e mettere 5 g di foglie in ogni provetta da centrifuga da 50 ml. Per raccogliere l'AWF, posizionare le foglie arrotolate con la punta rivolta verso l'alto in tubi con filo a rete fissato dai tappi dei tubi.</li>
		<li>Centrifugare a 4 °C, 500 x g per 5 min. Rimuovere le foglie e raccogliere l'AWF sul fondo della provetta utilizzando una pipetta. Da ciascuna provetta si possono ottenere circa 5 mL di AWF. NOTA: Al fine di ridurre al minimo l'interferenza delle proteine intracellulari e di estrarre la massima quantità di proteine AWF, l'estrazione può essere ripetuta 2x-3x, ma non più di 3x.</li>
	</ol></li>
	<li>Estrazione totale di proteine solubili<ol><li>Macinare 20 g di foglie in polvere fine in azoto liquido. Aggiungere il tampone di estrazione (50 mM Tris-HCl pH 7,4, 150 mM NaCl, 10% glicerolo, 1% Triton X-100, 0,034% β-mercaptoetanolo (vedere la Tabella dei materiali)) in rapporto 1: 2.</li>
		<li>Agitare l'omogeneizzato in un mixer verticale per 30 minuti a 4 °C. Centrifugare omogeneizzare a 13.000 x g per 15 min a 4 °C.</li>
		<li>Trasferire il surnatante in una nuova provetta e ripetere la centrifugazione per 15 minuti. Trasferire il surnatante chiarificato in un'altra provetta.</li>
		<li>Filtrare il surnatante attraverso un filtro da 0,22 μm (vedi Tabella dei materiali) per ottenere l'estratto proteico solubile totale.</li>
	</ol></li>
</ol>  
6. Purificazione di GFP-His con cromatografia di affinità al nichel (Ni)
NOTA: Eseguire tutti i passaggi a 4 °C.
<ol><li>Aggiungere la resina di Ni sefarosio excel (vedere la tabella dei materiali) a una provetta in un rapporto 1:1000 rispetto al volume dell'estratto proteico del passaggio 5.1.6. Il volume della resina è pari a 1/1000del volume dell'estratto proteico.</li>
	<li>Lavare separatamente la resina Ni 3x con un volume di resina 10x di ddH2O e PB (pH 7,0) separatamente e rimuovere il surnatante.</li>
	<li>Incubare l'estratto proteico con la resina di Ni per 2 ore per consentire il legame completo. Centrifugare a 500 x g per 5 minuti per rimuovere le proteine non legate nel surnatante. Lavare 3 volte con 20 volte il volume di resina di PB. Centrifugare a 500 x g per 5 minuti e rimuovere il surnatante.</li>
	<li>Eluire GFP-His aggiungendo 10 volte il volume di resina di 250 mM di imidazolo (50 mM di Tris-HCl pH 7,4, 150 mM di NaCl, 10% di glicerolo, 250 mM di imidazolo) e raccogliere il surnatante.</li>
</ol>7. Colorazione blu di Coomassie delle proteine
<ol><li>Aggiungere 10 μL di tampone di caricamento SDS 5x a 40 μL di campioni proteici, far bollire per 10 minuti, quindi eseguire su SDS-PAGE al 15% a 110 V per 60 minuti.</li>
	<li>Colorare i gel proteici con soluzione di blu di Coomassie (0,1% R-250, 25% isopropanolo, 10% acido acetico glaciale) per 2 ore.</li>
	<li>Versare la soluzione e lavare con soluzione decolorante per 2 ore. Osservare il gel; Le proteine sul gel saranno colorate di blu.</li>
</ol>8. Western blotting delle proteine
<ol><li>Aggiungere 10 μL di tampone di caricamento SDS 5x a 40 μL di campioni proteici, far bollire per 10 minuti, quindi eseguire su SDS-PAGE al 15% a 110 V per 60 minuti.</li>
	<li>Trasferire le proteine su una membrana di nitrocellulosa da 0,45 μm a 280 mA. Bloccare la membrana con latte scremato al 5% per 1 ora.</li>
	<li>Diluire gli anticorpi anti-His-tag a 1:5000 e incubare la membrana con gli anticorpi per 1 ora. Utilizzare la soluzione salina tris-tamponata e tween 20 (TBST) per lavare la membrana 4 volte per 5 minuti.</li>
	<li>Diluire gli anticorpi anti-topo a 1:10000 e incubare la membrana con gli anticorpi per 1 ora. Utilizzare TBST per lavare la membrana 4 volte per 5 minuti.</li>
	<li>Mescolare il rivelatore (vedi Tabella dei materiali) nel rapporto di 1:1. Aggiungere 400 μl alla membrana e lasciare agire per 1 minuto. Scatta fotografie dopo 1 minuto di esposizione con un visualizzatore.</li>
</ol>9. Saggio della concentrazione proteica
<ol><li>Aggiungere 5 μL di proteine a 1 mL di reagente colorante Bradford 1x e reagire per 5 minuti.</li>
	<li>Determinare il valore OD595 dopo l'azzeramento e calcolare la concentrazione proteica dalla curva standard.</li>
</ol>10. Quantificazione delle proteine 31
<ol><li>Eseguire il Western blotting di campioni proteici insieme agli standard.</li>
	<li>Esegui la quantificazione della GFP in rapporto agli standard utilizzando l'analisi della scala di grigi ImageJ 1.5.0.</li>
</ol>

Estrazione e purificazione efficiente di GFP-His dalle foglie di N. benthamiana

<ol>
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(94), e52113 (2014).</li><li>Nandi, 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=Process+development+and+economic+evaluation+of+recombinant+human+lactoferrin+expressed+in+rice+grain.">Process development and economic evaluation of recombinant human lactoferrin expressed in rice grain.</a> Transgenic Res. 14 (3), 237-249 (2005).</li><li>Menkhaus, T., 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=Considerations+for+the+recovery+of+recombinant+proteins+from+plants.">Considerations for the recovery of recombinant proteins from plants.</a> Biotechnol Prog. 20 (4), 1001-1014 (2004).</li><li>Streatfield, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approaches+to+achieve+high-level+heterologous+protein+production+in+plants.">Approaches to achieve high-level heterologous protein production in plants.</a> Plant Biotechnol J. 5 (1), 2-15 (2007).</li><li>Yamamoto, T., 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=Improvement+of+the+transient+expression+system+for+production+of+recombinant+proteins+in+plants.">Improvement of the transient expression system for production of recombinant proteins in plants.</a> Sci Rep. 8 (1), 4755 (2018).</li><li>Zhao, J., 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=Wheat+Apoplast-localized+lipid+transfer+protein+TaLTP3+enhances+defense+responses+against+Puccinia+triticina.">Wheat Apoplast-localized lipid transfer protein TaLTP3 enhances defense responses against Puccinia triticina.</a> Front Plant Sci. 12, 771806 (2021).</li><li>Venkataraman, 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=Recent+advances+in+expression+and+purification+strategies+for+plant+made+vaccines.">Recent advances in expression and purification strategies for plant made vaccines.</a> Front Plant Sci. 14, 1273958 (2023).</li><li>Witzel, K., Shahzad, M., Matros, A., Mock, H. P., M&#252;hling, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+evaluation+of+extraction+methods+for+apoplastic+proteins+from+maize+leaves.">Comparative evaluation of extraction methods for apoplastic proteins from maize leaves.</a> Plant Meth. 7, 48 (2011).</li></ol>

Gli autori dichiarano di non avere conflitti di interesse.

L'uso delle piante per produrre proteine terapeutiche si è espanso rapidamente negli ultimi anni 1,2,3,4,5,6. I geni codificanti le proteine vengono clonati in vettori di espressione e trasportati nei tessuti vegetali tramite agroinfiltrazione. Dopo la produzione da parte delle cellule vegetali, le proteine vengono purificat...

Al giorno d'oggi, sono in fase di studio varie proteine terapeutiche ricombinanti prodotte dalle piante, tra cui anticorpi, vaccini, proteine bioattive, enzimi e piccoli polipeptidi 1,2,3,4,5,6. Le piante stanno diventando una piattaforma sempre più utilizzata per la produzione di proteine terapeutiche grazie alla loro sicurezza, al basso costo e alla capacità di scalare rapidamente la produzione 7,8. L'espressione e la modificazione delle proteine nei sistemi vegetali, insieme alla purificazione delle proteine, sono fattori critici che determinano la produttività dei bioreattori delle piante 9,10. Migliorare la produttività delle piante e ottenere proteine target di elevata purezza sono le sfide principali che i sistemi di produzione a base vegetale devono affrontare11,12.
La localizzazione subcellulare e la modifica delle proteine ricombinanti dipendono dallo specifico peptide segnale codificato all'N-terminale, che indirizza la proteina alla sua destinazione finale nell'organello durante l'espressione genica. Le proteine ricombinanti sono progettate per localizzarsi nell'apoplasto, nel reticolo endoplasmatico (ER), nel cloroplasto, nel vacuolo o nel citoplasma, in base alle loro proprietà uniche13. Per gli antigeni e gli anticorpi, sono preferite le proteine secrete. Prima della secrezione, le proteine progrediscono attraverso l'ER e il Golgi per il corretto ripiegamento e le modifiche post-traduzionali 14,15,16.
Dopo la produzione nelle piante, le proteine ricombinanti devono essere purificate da estratti vegetali. Tipicamente, le proteine vengono purificate da estratti vegetali solubili totali, che contengono abbondanti proteine intracellulari, prodotti mal ripiegati e non modificati e citocromi17. Per rimuovere le impurità, gli estratti vegetali subiscono un frazionamento esteso, tra cui la cromatografia di affinità specifica per ribulosio-1,5-bisfosfato carbossilasi/ossigenasi (RuBisCO), la precipitazione dei fitati, la precipitazione del polietilenglicole e la regolazione della temperatura e del pH18,19. Tali passaggi di rimozione delle impurità sono laboriosi e possono compromettere la qualità delle proteine.
Il compartimento cellulare vegetale oltre la membrana plasmatica è definito come apoplasto, che comprende la parete cellulare, gli spazi intercellulari e lo xilema20. La fase acquosa è comunemente chiamata fluido di lavaggio apoplast (AWF). L'AWF contiene molecole secrete dalle cellule per regolare numerosi progressi biologici come il trasporto, il metabolismo della parete cellulare e le risposte allo stress21,22. A differenza del TSP, i costituenti molecolari dell'AWF sono meno complessi. L'estrazione di proteine ricombinanti da AWF può evitare la contaminazione da proteine intracellulari, prodotti mal ripiegati e residui citoplasmatici. In breve, il lipide di estrazione entra nelle foglie attraverso gli stomi a pressione negativa e l'AWF viene raccolto mediante centrifugazione delicata23. Sebbene l'isolamento dell'AWF sia ampiamente utilizzato per studiare il progresso biologico all'interno dell'apoplasto 24,25,26,27, non è stato sfruttato nei sistemi di produzione a base vegetale.
Migliorare la produttività delle piante e ottenere proteine target ad alta purezza sono sfide centrali per i sistemi di produzione vegetale28. Tipicamente, le proteine vengono purificate da estratti vegetali solubili totali contenenti grandi quantità di proteine intracellulari, prodotti mal ripiegati e non modificati e citocromi29, che richiedono grandi costi per la successiva purificazione30. L'uso di metodi di estrazione delle proteine AWF per l'estrazione di proteine ricombinanti esogene secrete nell'apoplast può migliorare efficacemente l'omogeneità delle proteine ricombinanti e ridurre il costo della purificazione delle proteine. Qui, utilizziamo un peptide segnale PR1a per abilitare la secrezione di proteine esogene nello spazio dell'apoplasto e introduciamo un metodo dettagliato per la purificazione di proteine ricombinanti dall'AWF di N. benthamiana.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 mesh nylon fine mesh yarn</td>
			<td>Guangzhou Huayu Trading limited company</td>
			<td>hy-230724-2</td>
			<td>For AWF protein extraction</td>
		</tr>
		<tr>
			<td>50 ml centrifuge tubes</td>
			<td>Vazyme</td>
			<td>TCF00150</td>
			<td>For centrifuge</td>
		</tr>
		<tr>
			<td>ClonExpress II one step cloning kit</td>
			<td>Vazyme</td>
			<td>C112-02</td>
			<td>For clone</td>
		</tr>
		<tr>
			<td>FastDigest Sac1</td>
			<td>NEB</td>
			<td>R3156S</td>
			<td>For clone</td>
		</tr>
		<tr>
			<td>FastDigest XbaI</td>
			<td>NEB</td>
			<td>FD0685</td>
			<td>For clone</td>
		</tr>
		<tr>
			<td>ImageJ 1.5.0</td>
			<td>/</td>
			<td>/</td>
			<td>For greyscale analysis</td>
		</tr>
		<tr>
			<td>Large filter</td>
			<td>Thermo Fisher</td>
			<td>NEST</td>
			<td>For filtering</td>
		</tr>
		<tr>
			<td>Laser scanning confocal microscopy</td>
			<td>Leica SP8</td>
			<td></td>
			<td>For subcellular localization</td>
		</tr>
		<tr>
			<td>Ni sepharose excel</td>
			<td>Cytiva</td>
			<td>10339806</td>
			<td>For protein purification</td>
		</tr>
		<tr>
			<td>Precast protein plus gel</td>
			<td>YEASEN</td>
			<td>36246ES10</td>
			<td>For SDS-PAGE</td>
		</tr>
		<tr>
			<td>Small filter</td>
			<td>Nalgene</td>
			<td>1660045</td>
			<td>For filtering</td>
		</tr>
		<tr>
			<td>SuperSignal West Femto Substrate Trial Kit</td>
			<td>Thermo Fisher</td>
			<td>34076</td>
			<td>For western blotting</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>SCR</td>
			<td>30188928</td>
			<td>To configure the extraction buffer</td>
		</tr>
		<tr>
			<td>Type rotaryvang vacuum pump</td>
			<td>Zhejiang taizhouqiujing vacuum pump limited company</td>
			<td>2XZ-2</td>
			<td>For vacuum infiltration</td>
		</tr>
		<tr>
			<td>Vertical mixer</td>
			<td>Haimen Qilinbel Instrument Manufacturing limited company</td>
			<td>BE-1200</td>
			<td>For mixing</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>SIGMA</td>
			<td>BCBK8223V</td>
			<td>To configure the extraction buffer</td>
		</tr>
	</tbody>
</table>

apoplast-extraction based method to improve the purity of plant produced recombinant protein  

Le piante sono un sistema di espressione eucariotica in via di sviluppo che viene esplorato per produrre proteine terapeutiche. La purificazione delle proteine ricombinanti dalle piante è una delle fasi più critiche del processo di produzione. In genere, le proteine sono state purificate da proteine solubili totali (TSP) e la presenza di varie proteine intracellulari e citocromi pone sfide per le successive fasi di purificazione delle proteine. Inoltre, la maggior parte delle proteine terapeutiche come antigeni e anticorpi vengono secrete per ottenere una corretta glicosilazione e la presenza di proteine non completamente modificate porta a strutture incoerenti di antigeni o anticorpi. Questo lavoro introduce un metodo più efficace per ottenere proteine ricombinanti altamente purificate dallo spazio apoplastico delle piante. La proteina fluorescente verde ricombinante (GFP) è ingegnerizzata per essere secreta nell'apoplasto di Nicotiana benthamiana e viene quindi estratta utilizzando un metodo di infiltrazione-centrifugazione. La GFP-His dell'apoplasto estratto viene quindi purificata mediante cromatografia di affinità con nichel. A differenza dei metodi tradizionali del TSP, la purificazione dall'apoplast produce proteine ricombinanti altamente purificate. Questo rappresenta un importante miglioramento tecnologico per i sistemi di produzione degli impianti.

GFP-His è bersaglio della secrezione nello spazio apoplastico di N. benthamiana La GFP è stata clonata nel plasmide pCAMBIA1300 con un peptide segnale PR1a N-terminale per la secrezione e un His-tag C-terminale per la purificazione, generando p35s-PR1a-GFP-His (Figura 1A). La proteina ricombinante è stata espressa transitoriamente in N. benthamiana e una banda di 30 kDa è stata rilevata mediante western blot utilizzando anticorpi ant...

Autore in primo piano: Ottimizzazione dell'estrazione delle proteine apoplast per un recupero efficiente delle proteine ricombinanti dalle cellule vegetali

La purificazione delle proteine ricombinanti dai sistemi vegetali è solitamente messa alla prova dalle proteine vegetali. Questo lavoro fornisce un metodo per estrarre e purificare efficacemente una proteina ricombinante secreta dall'apoplasto di Nicotiana benthamiana.

Metodo basato sull'estrazione di Apoplast per migliorare la purezza delle proteine ricombinanti prodotte dalle piante  

Plants are a newly developing eukaryotic expression system being explored to produce therapeutic proteins. Purification of recombinant proteins from ...

apoplast-extraction-based-method-to-improve-purity-plant-produced

Research

JoVE Journal

Biology

Apoplast-Extraction Based Method to Improve the Purity of Plant Produced Recombinant Protein  

844 Views.  Shandong First Medical University. Abbiamo studiato come estrarre le proteine ricombinanti dalle cellule vegetali in modo più efficiente. Abbiamo risposto alla domanda se il metodo di estrazione della proteina apoplast sia fattibile quando applicato alle proteine ricombinanti. Questo metodo di estrazione estrae le proteine secrete nell'apoplasto, in modo che abbiano omogeneità.Non è necessaria la disgregazione cellulare, il che riduce l'interferenza delle proteine intracellulari e i ...

Video: Autore in primo piano: Ottimizzazione dell'estrazione delle proteine apoplast per un recupero efficiente delle proteine ricombinanti dalle cellule vegetali

A Method For Production of Recombinant mCD1d Protein in Insect Cells.

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated heavy chains that are noncovalently associated with beta2-microglobulin. They bind lipids rather than peptides. Although their structure confirms the similarity of CD1 proteins to MHC class I and class II antigen presenting molecules, the mCD1d groove is relatively narrow, deep, and highly hydrophobic and it has two binding pockets instead of the several shallow pockets described for the classical MHC-encoded antigen-presenting molecules. Based upon their amino acid sequences, such a hydrobphobic groove provides an ideal environment for the binding of lipid antigens. The Natural Killer T (NKT) cells use their TCR to recognize glycolipids bound to or presented by CD1d. T cells reactive to lipids presented by CD1 have been involved in the protection against autoimmune and infectious diseases and in tumor rejection. Thus, the ability to identify, purify , and track the response of CD1-reactive NKT cell is of great importance . The generation of tetramers of alpha Galactosyl ceramide (a-Galcer) with CD1d has significant insight into the biology of NKT cells. Tetramers constructed from other CD1 molecules have also been generated and these new reagents have greatly expanded the knowledge of the functions of lipid-reactive T cells, with potential use in monitoring the response to lipid-based vaccines and in the diagnosis of autoimmune diseases and other treatments.

A Method to prepare Insect cells and infect them with baculovirus for the the purpose of production of recombinant mCD1d proteinand generating mCD1d tetramers.

Un metodo per la produzione di proteine ​​ricombinanti mCD1d in cellule di insetto.

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated ...

Ricerca

Biologia

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Utilizzando il sistema GELFREE 8100 Frazionamento per peso molecolare basata su Frazionamento con recupero della fase liquida

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

A PCR-based metodo di genotipizzazione di distinguere tra le varietà selvatiche di tipo ornamentale e di Imperata cylindrica</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

Un protocollo per l&#39;identificazione di interazioni proteina-proteina Sulla base di<sup&gt; 15</sup&gt; N marcatura metabolica, immunoprecipitazione, Spettrometria di massa quantitativa e di affinità di modulazione

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

Identificazione delle modificazioni post-traduzionali di proteine ​​vegetali Complessi

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

High Throughput Screening espressione quantitativa e purificazione Applicata al ricombinante Disulfide ricchi di Venom proteine ​​prodotte in<em&gt; E. coli</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Green Fluorescent Protein-base Espressione Proiezione di proteine ​​di membrana in<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the flexible, low-cost production of high-quality, bioactive products.
In this study, we compared the expression of a target human recombinant protein in traditional fermenter-based cell cultures (bacterial and insect) with plant-based expression systems, both transient and stable.
For each platform, we described the set-up, optimization and length of the production process, the final product quality and the yields and we evaluated provisional production costs, specific for the selected target recombinant protein.
Overall, our results indicate that bacteria are unsuitable for the production of the target protein due to its accumulation within insoluble inclusion bodies. On the other hand, plant-based systems are versatile platforms that allow the production of the selected protein at lower-costs than Baculovirus/insect cell system. In particular, stable transgenic lines displayed the highest-yield of the final product and transient expressing plants the fastest process development. However, not all recombinant proteins may benefit from plant-based systems but the best production platform should be determined empirically with a case-by-case approach, as described here.

In this study the expression of a target human recombinant protein in different production platforms was compared. We focused on traditional fermenter-based cultures and on plants, describing the set-up of each system and highlighting, on the basis of the reported results, the inherent limits and advantages for each platform.

Un&#39;analisi comparativa di espressione della proteina ricombinante in diversi biofabbriche: batteri, cellule di insetto e impianti

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the ...

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

Metodo standardizzato per la sterilizzazione di High-throughput di semi di Arabidopsis

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...

Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection

Rice stripe virus (RSV), which causes significant economic loss of agriculture in East Asia, entirely depends on insect vectors for its effective transmission among host rice. Laodelphax striatellus (small brown planthopper, SBPH) is the primary insect vector that horizontally transmits RSV while sucking sap from the phloem. Saliva plays a significant role in insects' feeding behavior. A convenient method that will be useful for research on insects' saliva with piercing-sucking feeding behavior is described here. In this method, insects were allowed to feed on an artificial diet sandwiched between two stretched paraffin film layers. The diet containing the saliva was collected each day, filtered, and concentrated for further analysis. Finally, the quality of collected saliva was examined by protein staining and immunoblotting. This method was exemplified by detecting the presence of RSV and a mucin-like protein in the saliva of SBPH. These artificial feeding and saliva collection method will lay a foundation for further research on factors in insect saliva related to feeding behavior and virus transmission.

Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection

The present protocol describes a method to collect sufficient saliva from piercing-sucking insects using an artificial medium. This is a convenient method for collecting insect saliva and studying salivary function on insect feeding behavior and vector-borne virus transmission.