Diese Arbeit wird unterstützt von der Youth Innovation Promotion Association CAS (2021084) und dem Key Deployment Project Support Fund des Dreijahresaktionsplans des Instituts für Mikrobiologie der Chinesischen Akademie der Wissenschaften.

1. Vorbereitung der Pflanzen von N. benthamiana 
<ol><li>Legen Sie die Sämlingsblöcke in eine Schale, fügen Sie 1 l Wasser hinzu und weichen Sie sie ca. 2 h lang ein, bis sie vollständig gesättigt sind.</li>
	<li>Streuen Sie die Wildtyp-Samen von N. benthamiana gleichmäßig auf die Sämlingsblöcke, bedecken Sie sie mit einem Deckel und keimen Sie 1 Woche lang. Anbauen Sie N. benthamiana in einem Gewächshaus mit 18 h Photoperiode, 24 °C, 45% relativer Luftfeuchtigkeit und düngen Sie alle 2 Wochen.</li>
	<li>Sobald die Samen gekeimt sind, entferne den Sämling vorsichtig mit einer Pinzette und achte darauf, dass seine Wurzeln nicht beschädigt werden. Grabe eine kleine Grube in der Mitte einer neuen Schale, setze den Sämling hinein und bedecke die Wurzeln leicht mit Erde. Mit einem Deckel abdecken und weiter wachsen.</li>
	<li>Wachsen Sie, bis die Sämlinge drei Blätter haben, mit geschlossenem Deckel. Nehmen Sie den Deckel ab, wenn das 4. wahre Blatt herauskommt.</li>
	<li>Lassen Sie die Pflanzen noch zwei Wochen weiterwachsen, bevor Sie die Agroinfiltration fortsetzen.</li>
</ol>2. Aufbau des rekombinanten GFP-His
<ol><li>Durchführung der N . benthamiana-Codon-Optimierung und Synthese von GFP und PR1a (MGFVLFSQLPSFLLVSTLLLLLFLVISHSCRAG) basierend auf den Gensequenzen.</li>
	<li>Führen Sie einen doppelten Aufschluss des pCAMBIA1300-Vektors mit den Enzymen Xba I und Sac I durch (siehe Materialtabelle).</li>
	<li>Durchführung der Amplifikation von PR1a unter Verwendung von Upstream-Primern (TTGGAGAGAACGGGGGACTCTAGAATGGGATT TGTTCTCTTTTC) und nachgeschaltete Primer (TCCTCG CCCTTGCTCACCATGTCGACCCCGGCACGGCAAGA GTGGG), Amplifikation von GFP unter Verwendung von Upstream-Primern (CCCACTCTTGCCGTGCCGGGGTCGACATGGTGAG CAAGGGCGAGGA) und nachgeschaltete Primer (GGGGAAATTCGAGCTCTCAGTGGTGATGGTGGTG GTGAGATCTTCCGGACTTGT).</li>
	<li>Unter Verwendung der homologen Rekombination klonieren PR1a und GFP in den pflanzlichen Expressionsvektor pCAMBIA1300. Nach der Übertragung in E. coli wählen Sie einzelne Klone aus und senden Sie sie zur Sequenzierung an das Unternehmen. Vergleich der Sequenzierungsergebnisse und des richtigen Plasmids ist das rekombinante Plasmid pCAMBIA1300-PR1a-GFP-His, das als p35s-PR1a-GFP-His bezeichnet wird.</li>
</ol>3. Produktion von GFP-His in N. benthamiana
<ol><li>1 μl GFP-Plasmid in 50 μl Agrobacterium-Rezeptorzellen GV3101 geben, vorsichtig mischen und in den Elektrodenbecher geben, nach Stromschlag am Elektroschocker entfernen.</li>
	<li>Verteilen Sie transformiertes Agrobacterium tumefaciens auf eine LB-Platte mit festem Medium mit 50 μg/ml Kanamycin (Kan) und 50 μg/ml Rifampicin (Rif). Invertiert bei 28 °C 48 h inkubieren. Der Vektor pCAMBIA1300 ist kan-resistent und Agrobacterium ist rif-resistent; Nur das richtige Agrobacterium kann auf Platten mit kan und rif wachsen. Bei den monoklonalen Klonen handelte es sich um positive Transformanten mit dem Namen A. tumefaciens GV3101-p35s-PR1a-GFP-His (AtGPG).</li>
	<li>Verwenden Sie einen sterilisierten Zahnstocher, um eine angemessene Menge AtGPG aufzunehmen und es mit Kan und Rif in 4 ml flüssiges LB zu impfen. Kultur bei 28 °C, 220-300 U/min für 24 h.</li>
	<li>Subkulturieren Sie Bakterien 1:50 in frisches LB (50 μg/mL Kan, 10 mM 2-(N-Morpholino) Ethansulfonsäure (MES), 20 μM Acetosyringon (AS)) und inkubieren bei 28 °C, 220-300 U/min für 10-12 h.</li>
	<li>Ernten Sie die Bakterien durch Zentrifugation bei 1500 x g für 10 min und resuspendieren Sie das Pellet mit 1 mL MMA (10 mM MgCl2, 10 mM MES pH 5,6, 100 μM AS).</li>
	<li>Messen und justieren Sie die optische Dichte (OD) von AtGPG aufOD 600=0,8 mit MMA. Inkubieren Sie die Bakterien für weitere 2-4 Stunden bei Raumtemperatur. Ziehen Sie die Bakterienlösung mit einer nadellosen Spritze auf, drücken Sie sie leicht gegen die Rückseite der Blätter und üben Sie Druck aus, um die Lösung in die Blätter einzudringen. Injizieren Sie nur die mittleren 3-4 Blätter pro Pflanze.</li>
	<li>Bauen Sie N. benthamiana 3-4 Tage lang im Gewächshaus an, bevor Sie die Blätter für die Proteinextraktion ernten.</li>
</ol>4. Beobachtung der subzellulären Lokalisation von GFP
<ol><li>Nach 48 h Agroinfiltration wählen Sie ein flaches Blatt und verwenden Sie einen 6 mm Locher, um die Klinge zum Stanzen einzuspannen.</li>
	<li>30%ige Saccharoselösung auf einen Objektträger träufeln, die Blattprobe mit der Rückseite nach oben platzieren und mit einem Deckglas abdecken.</li>
	<li>Stellen Sie die Anregungswellenlänge auf 488 nm ein und detektieren Sie die grüne Fluoreszenz bei 490-550 nm nach 400-facher Vergrößerung. Machen Sie das erste Foto, sobald die Folie vorbereitet ist, und ein weiteres 10 Minuten später.</li>
</ol>5. Extraktion von GFP aus der Pflanze
<ol><li>AWF-Extraktion<ol><li>Sammeln Sie die intakten frischen Blätter von N. benthamiana , die 3 Tage nach der Agroinfiltration rekombinantes GFP exprimieren. Behandeln Sie das Blatt schonend und bewahren Sie die Integrität des Blattes, um einen intrazellulären Proteinausfluss zu vermeiden. Verwenden Sie gesammelte Blätter sofort für den nächsten Schritt, um einen Proteinabbau zu vermeiden.</li>
		<li>Spülen Sie die Blattklinge mit vorgekühltem, sterilisiertem ddH2O ab, um Schmutz zu entfernen.</li>
		<li>Frische Blätter werden gewogen und 20 g Blätter (mit der abaxialen Seite nach unten) in 100 mL vorgekühlten 100 mM Phosphatpuffer (PB 100 mM Na2, HPO4 und 100 mM NaH2PO4 wurden separat konfiguriert und in einem Verhältnis von 2,2:1 und pH 7,0 gemischt) in ein 200 mL Weithalsbecherglas getaucht. Die Blätter mit 100er feinem Nylongarn (siehe Materialtabelle) und einer Kunststoffplatte abdecken. HINWEIS: Die Vorkühlung verhindert den Proteinabbau. Die Puffervolumina können basierend auf der experimentellen Skala angepasst werden. Stellen Sie sicher, dass alle Blätter eingetaucht sind.</li>
		<li>Setzen Sie das Becherglas in eine Vakuumpumpe ein. Wenden Sie 1 Minute lang ein Vakuum von 0,8 MPa an und stellen Sie es dann schnell wieder auf Atmosphärendruck her.</li>
		<li>Entfernen Sie die Blätter aus dem Puffer und tupfen Sie sie vorsichtig mit saugfähigem Papier trocken. Die Blätter aufrollen, mit Netzgarn umwickeln und 5 g Blätter in jedes 50-ml-Zentrifugenröhrchen geben. Um AWF zu sammeln, legen Sie die gerollten Blätter mit der Spitze nach oben in Röhren mit Netzgarn, das an den Röhrenkappen befestigt ist.</li>
		<li>Zentrifugieren Sie bei 4 °C, 500 x g für 5 min. Entfernen Sie die Blätter und sammeln Sie das AWF mit einer Pipette am Boden des Röhrchens. Aus jedem Röhrchen können etwa 5 mL AWF gewonnen werden. HINWEIS: Um die Interferenz durch intrazelluläre Proteine zu minimieren und die maximale Menge an AWF-Proteinen zu extrahieren, kann die Extraktion 2x-3x, aber nicht mehr als 3x wiederholt werden.</li>
	</ol></li>
	<li>Extraktion des gesamten löslichen Proteins<ol><li>20 g Blätter in flüssigem Stickstoff zu feinem Pulver mahlen. Geben Sie Extraktionspuffer (50 mM Tris-HCl pH 7,4, 150 mM NaCl, 10 % Glycerin, 1 % Triton X-100, 0,034 % β-Mercaptoethanol (siehe Materialtabelle)) im Verhältnis 1: 2 zu.</li>
		<li>Das Homogenat in einem vertikalen Mischer 30 Minuten lang bei 4 °C schütteln. Zentrifugieren Sie das Homogenat bei 13.000 x g für 15 min bei 4 °C.</li>
		<li>Den Überstand in ein neues Röhrchen umfüllen und die Zentrifugation 15 Minuten wiederholen. Den gereinigten Überstand in ein anderes Röhrchen umfüllen.</li>
		<li>Filtrieren Sie den Überstand durch einen 0,22-μm-Filter (siehe Materialtabelle), um den gesamten löslichen Proteinextrakt zu erhalten.</li>
	</ol></li>
</ol>  
6. Reinigung von GFP-His mit Nickel (Ni)-Affinitätschromatographie
HINWEIS: Führen Sie alle Schritte bei 4 °C durch.
<ol><li>Geben Sie Ni-Sepharose-Excel-Harz (siehe Materialtabelle) in einem Verhältnis von 1:1000 in ein Röhrchen relativ zum Proteinextraktvolumen aus Schritt 5.1.6. Das Harzvolumen entspricht 1/1000des Volumens des Proteinextrakts.</li>
	<li>Ni-Harz 3x mit 10x Harzvolumen von ddH2O und PB (pH 7,0) nacheinander getrennt waschen und den Überstand entfernen.</li>
	<li>Inkubieren Sie den Proteinextrakt 2 Stunden lang mit Ni-Harz, um eine vollständige Bindung zu ermöglichen. Bei 500 x g für 5 min zentrifugieren, um ungebundene Proteine im Überstand zu entfernen. 3x mit 20x Harz Volumen PB waschen. Bei 500 x g 5 min zentrifugieren und den Überstand entfernen.</li>
	<li>Eluieren Sie GFP-His durch Zugabe von 10x Harzvolumen von 250 mM Imidazol (50 mM Tris-HCl pH 7,4, 150 mM NaCl, 10% Glycerin, 250 mM Imidazol) und sammeln Sie den Überstand.</li>
</ol>7. Coomassie-Blau-Färbung von Proteinen
<ol><li>10 μl 5x SDS-Ladepuffer zu 40 μl Proteinproben geben, 10 min kochen lassen, dann 60 min lang mit 15 % SDS-PAGE bei 110 V laufen lassen.</li>
	<li>Färben Sie Proteingele mit Coomassieblau-Lösung (0,1 % R-250, 25 % Isopropanol, 10 % Eisessig) für 2 h.</li>
	<li>Gießen Sie die Lösung ab und waschen Sie sie 2 h lang mit Entfärbungslösung. Beobachten Sie das Gel; Proteine auf dem Gel werden blau gefärbt.</li>
</ol>8. Western Blotting von Proteinen
<ol><li>10 μl 5x SDS-Ladepuffer zu 40 μl Proteinproben geben, 10 min kochen lassen, dann 60 min lang mit 15 % SDS-PAGE bei 110 V laufen lassen.</li>
	<li>Übertragen Sie Proteine auf eine 0,45 μm große Nitrozellulosemembran bei 280 mA. Blockieren Sie die Membran 1 h lang mit 5 % Magermilch.</li>
	<li>Verdünnen Sie die Anti-His-Tag-Antikörper bei 1:5000 und inkubieren Sie die Membran mit den Antikörpern 1 h lang. Mit der Tris-gepufferten Kochsalzlösung und dem Tween 20 (TBST) die Membran 4x für 5 min waschen.</li>
	<li>Verdünnen Sie die Anti-Maus-Antikörper bei 1:10000 und inkubieren Sie die Membran mit den Antikörpern 1 h lang. Waschen Sie die Membran mit TBST 4x für 5 min.</li>
	<li>Den Entwickler (siehe Materialtabelle) im Verhältnis 1:1 mischen. 400 μl auf die Membran geben und 1 min reagieren lassen. Machen Sie Fotos nach 1 Minute Belichtung mit einem Visualizer.</li>
</ol>9. Proteinkonzentrations-Assay
<ol><li>Geben Sie 5 μl Protein zu 1 ml Bradford 1x Farbstoffreagenz und reagieren Sie 5 Minuten lang.</li>
	<li>Bestimmen Sie den OD595-Wert nach dem Nullstellen und berechnen Sie die Proteinkonzentration aus der Normkurve.</li>
</ol>10. Quantifizierung von Proteinen 31
<ol><li>Durchführung von Western-Blotting von Proteinproben zusammen mit Standards.</li>
	<li>Führen Sie eine Quantifizierung des GFP nach dem Verhältnis zu Standards mit der Graustufenanalyse von ImageJ 1.5.0 durch.</li>
</ol>

Effiziente Extraktion und Reinigung von GFP-His aus N. benthamiana-Blättern

<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>

Die Autoren erklären, dass kein Interessenkonflikt besteht.

Die Verwendung von Pflanzen zur Herstellung therapeutischer Proteine hat in den letzten Jahren schnell zugenommen 1,2,3,4,5,6. Proteinkodierende Gene werden in Expressionsvektoren kloniert und über Agroinfiltration in pflanzliches Gewebe abgegeben. Nach der Produktion durch Pflanzenzellen werden Proteine für nachgelagerte A...

Heutzutage werden verschiedene pflanzlich produzierte rekombinante therapeutische Proteine untersucht, darunter Antikörper, Impfstoffe, bioaktive Proteine, Enzyme und kleine Polypeptide 1,2,3,4,5,6. Pflanzen werden aufgrund ihrer Sicherheit, ihrer geringen Kosten und ihrer Fähigkeit, die Produktion schnell zu skalieren, zu einer zunehmend genutzten Plattform für die Herstellung therapeutischer Proteine 7,8. Die Proteinexpression und -modifikation in pflanzlichen Systemen sind zusammen mit der Proteinreinigung kritische Faktoren, die die Produktivität von pflanzlichen Bioreaktoren bestimmen 9,10. Die Steigerung der Pflanzenproduktivität und die Gewinnung hochreiner Zielproteine sind zentrale Herausforderungen für pflanzenbasierte Produktionssysteme11,12.
Die subzelluläre Lokalisierung und Modifikation rekombinanter Proteine hängt von dem spezifischen Signalpeptid ab, das am N-Terminus kodiert ist und das Protein während der Genexpression zu seinem endgültigen Organellenziel führt. Rekombinante Proteine sind so konzipiert, dass sie aufgrund ihrer einzigartigen Eigenschaften im Apoplasten, im endoplasmatischen Retikulum (ER), im Chloroplasten, in der Vakuole oder im Zytoplasma lokalisiertsind 13. Bei Antigenen und Antikörpern werden sekretierte Proteine bevorzugt. Vor der Sekretion durchlaufen die Proteine ER und Golgi für eine korrekte Faltung und posttranslationale Modifikationen 14,15,16.
Nach der Produktion in Pflanzen müssen rekombinante Proteine aus Pflanzenextrakten gereinigt werden. Typischerweise werden Proteine aus vollständig löslichen Pflanzenextrakten gereinigt, die reichlich intrazelluläre Proteine, fehlgefaltete und unmodifizierte Produkte und Cytochrome enthalten17. Um Verunreinigungen zu entfernen, werden Pflanzenextrakte einer umfangreichen Fraktionierung unterzogen, einschließlich einer Ribulose-1,5-Bisphosphat-Carboxylase/Oxygenase (RuBisCO)-spezifischen Affinitätschromatographie, Phytatfällung, Polyethylenglykolfällung und Einstellung von Temperatur und pH18,19. Solche Schritte zur Entfernung von Verunreinigungen sind mühsam und können die Proteinqualität beeinträchtigen.
Das pflanzliche Zellkompartiment jenseits der Plasmamembran wird als Apoplast definiert und umfasst die Zellwand, die Interzellularräume und das Xylem20. Die wässrige Phase wird allgemein als Apoplast-Waschflüssigkeit (AWF) bezeichnet. AWF enthält Moleküle, die von Zellen sezerniert werden, um zahlreiche biologische Prozesse wie Transport, Zellwandstoffwechsel und Stressreaktionen zu regulieren21,22. Im Gegensatz zu TSP sind die molekularen Bestandteile von AWF weniger komplex. Die Extraktion rekombinanter Proteine aus AWF kann eine Kontamination durch intrazelluläre Proteine, fehlgefaltete Produkte und zytoplasmatische Überreste vermeiden. Kurz gesagt, das Extraktionslipid gelangt unter Unterdruck durch die Spaltöffnungen in die Blätter, und das AWF wird durch schonende Zentrifugationaufgefangen 23. Während die Isolierung von AWF weit verbreitet ist, um den biologischen Fortschritt in Apoplast 24,25,26,27 zu untersuchen, wurde sie in pflanzlichen Produktionssystemen nicht genutzt.
Die Verbesserung der Pflanzenproduktivität und die Gewinnung hochreiner Zielproteine sind zentrale Herausforderungen für pflanzliche Produktionssysteme28. Typischerweise werden Proteine aus vollständig löslichen Pflanzenextrakten gereinigt, die große Mengen an intrazellulären Proteinen, fehlgefalteten und unmodifizierten Produkten und Cytochromenenthalten 29, was hohe Kosten für die anschließende Reinigungerfordert 30. Die Verwendung von AWF-Proteinextraktionsmethoden zur Extraktion exogener rekombinanter Proteine, die in Apoplasten sezerniert werden, kann die Homogenität rekombinanter Proteine effektiv verbessern und die Kosten für die Proteinreinigung senken. Hier verwenden wir ein Signalpeptid PR1a, um eine exogene Proteinsekretion in den Apoplastenraum zu ermöglichen und stellen eine detaillierte Methode zur rekombinanten Proteinreinigung aus dem AWF von N. benthamiana vor.

<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  

Pflanzen sind ein neu entwickeltes eukaryotisches Expressionssystem, das zur Herstellung therapeutischer Proteine erforscht wird. Die Reinigung von rekombinanten Proteinen aus Pflanzen ist einer der kritischsten Schritte im Produktionsprozess. In der Regel wurden Proteine aus vollständig löslichen Proteinen (TSP) gereinigt, und das Vorhandensein verschiedener intrazellulärer Proteine und Cytochrome stellt eine Herausforderung für nachfolgende Proteinreinigungsschritte dar. Darüber hinaus werden die meisten therapeutischen Proteine wie Antigene und Antikörper sezerniert, um eine ordnungsgemäße Glykosylierung zu erhalten, und das Vorhandensein unvollständig modifizierter Proteine führt zu inkonsistenten Antigen- oder Antikörperstrukturen. In dieser Arbeit wird eine effektivere Methode zur Gewinnung hochreiner rekombinanter Proteine aus dem apoplastischen Pflanzenraum vorgestellt. Das rekombinante grün fluoreszierende Protein (GFP) wird so verändert, dass es in den Apoplasten von Nicotiana benthamiana sezerniert und dann mit einer Infiltrations-Zentrifugationsmethode extrahiert wird. Das GFP-His aus dem extrahierten Apoplasten wird dann durch Nickel-Affinitätschromatographie aufgereinigt. Im Gegensatz zu den traditionellen Methoden von TSP entstehen bei der Aufreinigung aus dem Apoplasten hochreine rekombinante Proteine. Dies stellt eine wichtige technologische Verbesserung für pflanzliche Produktionssysteme dar.

GFP-His ist für die Sekretion in den apoplastischen Raum von N. benthamiana bestimmt GFP wurde mit einem N-terminalen PR1a-Signalpeptid für die Sekretion und einem C-terminalen His-Tag für die Aufreinigung in das pCAMBIA1300-Plasmid kloniert, wodurch p35s-PR1a-GFP-His erzeugt wurde (Abbildung 1A). Das rekombinante Protein wurde transient in N. benthamiana exprimiert, und eine 30 kDa-Bande wurde 3 Tage nach der Agroinfiltration mittels...

Autor im Rampenlicht: Optimierung der Apoplast Proteinextraktion für die effiziente Rückgewinnung rekombinanter Proteine aus Pflanzenzellen

Die Aufreinigung rekombinanter Proteine aus pflanzlichen Systemen wird in der Regel durch pflanzliche Proteine herausgefordert. Diese Arbeit stellt eine Methode zur effektiven Extraktion und Reinigung eines sekretierten rekombinanten Proteins aus dem Apoplast von Nicotiana benthamiana dar.

Auf Apoplast-Extraktion basierende Methode zur Verbesserung der Reinheit von pflanzlich produzierten rekombinanten Proteinen  

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  

760 Aufrufe.  Shandong First Medical University. Wir haben untersucht, wie man rekombinante Proteine effizienter aus Pflanzenzellen extrahieren kann. Wir beantworteten die Frage, ob die Methode der Apoplast-Proteinextraktion praktikabel ist, wenn sie auf die rekombinanten Proteine angewendet wird. Bei dieser Extraktionsmethode werden Proteine extrahiert, die in den Apopen sezerniert werden, so dass sie Homogenität aufweisen.Es ist kein Zellaufschluss erforderlich, wodurch die Interferenz durch intra...

Serie: Autor im Rampenlicht: Optimierung der Apoplast Proteinextraktion für die effiziente Rückgewinnung rekombinanter Proteine aus Pflanzenzellen

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.

Ein Verfahren zur Herstellung von rekombinantem Protein in mCD1d Insektenzellen.

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

Forschung

Biologie

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.

Mit dem GELFREE 8100 Fraktionierung System für Molecular Weight-Based Fraktionierung mit Liquid Phase Wiederherstellung

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).

Ein PCR-basierte Genotypisierung Methode, zwischen Wildtyp-und Ziersorten Unterscheiden von 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.

Ein Protokoll für die Identifizierung von Protein-Protein-Wechselwirkungen Basierend auf<sup&gt; 15</sup&gt; N Metabolische Markierung, Immunpräzipitation, Quantitative Massenspektrometrie und Affinity Modulation

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.

Bezeichnung des Post-translationale Modifikationen von Pflanzenproteinkomplexe

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.

Hochdurchsatz-Screening Quantitative Expression und Aufreinigung rekombinanter zu Disulfid-reichen Proteine ​​Venom Produziert in Applied<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-basierte Screening Expression von Membranproteinen 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.

Eine vergleichende Analyse der Expression rekombinanter Proteine ​​in verschiedenen Biofabrik: Bakterien, Insektenzellen und Pflanzensysteme

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.

Standardisierte Methode für Hochdurchsatz-Sterilisation von Arabidopsis Samen

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.