Name: Författare i fokus: Optimera Apoplast-proteinextraktion för effektiv återvinning av rekombinanta proteiner från växtceller
Uploaded: 2024-07-05T14:20:00
Description: Plants are a newly developing eukaryotic expression system being explored to produce therapeutic proteins. Purification of recombinant proteins from ...

Detta arbete stöds av Youth Innovation Promotion Association CAS (2021084) och Key Deployment Project Support Fund i den treåriga handlingsplanen från Institute of Microbiology, Chinese Academy of Sciences.

1. Beredning av N. benthamiana-växter 
<ol><li>Lägg plantblock i en bricka, tillsätt 1 L vatten och blötlägg i cirka 2 timmar tills de är helt mättade.</li>
	<li>Strö jämnt frön av vildtyp N. benthamiana på plantblocken, täck med ett lock och gro i 1 vecka. Odla N. benthamiana i ett växthus med 18 timmars fotoperiod, 24 °C, 45 % relativ luftfuktighet och gödsla varannan vecka.</li>
	<li>När fröna har grott, använd en pincett...

Effektiv extraktion och rening av GFP-His från N. benthamiana löv

<ol>
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J., S&#233;guin, A., Couillard, J., Tr&#233;panier, S., Landry, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+III:+Randomized+observer-blind+trial+to+evaluate+lot-to-lot+consistency+of+a+new+plant-derived+quadrivalent+virus+like+particle+influenza+vaccine+in+adults+18-49+years+of+age.">Phase III: Randomized observer-blind trial to evaluate lot-to-lot consistency of a new plant-derived quadrivalent virus like particle influenza vaccine in adults 18-49 years of age.</a> Vaccine. 39 (10), 1528-1533 (2021).</li><li>Siriwattananon, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant-produced+receptor-binding+domain+of+SARS-CoV-2+elicits+potent+neutralizing+responses+in+mice+and+non-human+primates.">Plant-produced receptor-binding domain of SARS-CoV-2 elicits potent neutralizing responses in mice and non-human primates.</a> Front Plant Sci. 12, 682953 (2021).</li><li>Leblanc, Z., Waterhouse, P., Bally, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant-based+vaccines:+The+way+ahead.">Plant-based vaccines: The way ahead.</a> Viruses. 13 (1), 5 (2020).</li><li>Sethi, L., Kumari, K., Dey, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+plants+for+efficient+production+of+therapeutics.">Engineering of plants for efficient production of therapeutics.</a> Mol Biotechnol. 63 (12), 1125-1137 (2021).</li><li>Webster, D. E., Thomas, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-translational+modification+of+plant-made+foreign+proteins;+glycosylation+and+beyond.">Post-translational modification of plant-made foreign proteins; glycosylation and beyond.</a> Biotechnol Adv. 30 (2), 410-418 (2012).</li><li>Streatfield, S. J. <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>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>Ullrich, K. K., Hiss, M., Rensing, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Means+to+optimize+protein+expression+in+transgenic+plants.">Means to optimize protein expression in transgenic plants.</a> Curr Opin Biotechnol. 32, 61-67 (2015).</li><li>Su, H., 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=Plant-made+vaccines+against+viral+diseases+in+humans+and+farm+animals.">Plant-made vaccines against viral diseases in humans and farm animals.</a> Front Plant Sci. 14, 1170815 (2023).</li><li>Agrawal, G. K., Jwa, N. S., Lebrun, M. H., Job, D., Rakwal, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+secretome:+Unlocking+secrets+of+the+secreted+proteins.">Plant secretome: Unlocking secrets of the secreted proteins.</a> Proteomics. 10 (4), 799-827 (2010).</li><li>Kim, T., Gondr&#233;-Lewis, M. C., Arnaoutova, I., Loh, Y. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dense-core+secretory+granule+biogenesis.">Dense-core secretory granule biogenesis.</a> Physiology. 21, 124-133 (2006).</li><li>Menkhaus, T. J., Bai, Y., Zhang, C., Nikolov, Z. L., Glatz, C. E. <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>Kim, S. T., Cho, K. S., Jang, Y. S., Kang, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+electrophoretic+analysis+of+rice+proteins+by+polyethylene+glycol+fractionation+for+protein+arrays.">Two-dimensional electrophoretic analysis of rice proteins by polyethylene glycol fractionation for protein arrays.</a> Electrophoresis. 22 (10), 2103-2109 (2001).</li><li>Xi, 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=Polyethylene+glycol+fractionation+improved+detection+of+low-abundant+proteins+by+two-dimensional+electrophoresis+analysis+of+plant+proteome.">Polyethylene glycol fractionation improved detection of low-abundant proteins by two-dimensional electrophoresis analysis of plant proteome.</a> Phytochemistry. 67 (21), 2341-2348 (2006).</li><li>Dani, V., Simon, W. J., Duranti, M., Croy, R. R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+tobacco+leaf+apoplast+proteome+in+response+to+salt+stress.">Changes in the tobacco leaf apoplast proteome in response to salt stress.</a> Proteomics. 5 (3), 737-745 (2005).</li><li>Delaunois, B., 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=Uncovering+plant-pathogen+crosstalk+through+apoplastic+proteomic+studies.">Uncovering plant-pathogen crosstalk through apoplastic proteomic studies.</a> Front Plant Sci. 5, 249 (2014).</li><li>Andreasson, E., Abreha, K. B., Resj&#246;, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+apoplast.">Isolation of apoplast.</a> Methods Mol. Biol. 1511, 233-240 (2017).</li><li>Lohaus, G., Pennewiss, K., Sattelmacher, B., Hussmann, M., Hermann, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+the+infiltration-centrifugation+technique+appropriate+for+the+isolation+of+apoplastic+fluid?+A+critical+evaluation+with+different+plant+species.">Is the infiltration-centrifugation technique appropriate for the isolation of apoplastic fluid? A critical evaluation with different plant species.</a> Physiol Plant. 111 (4), 457-465 (2001).</li><li>Nouchi, I., 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=Overcoming+the+difficulties+in+collecting+apoplastic+fluid+from+rice+leaves+by+the+infiltration-centrifugation+method.">Overcoming the difficulties in collecting apoplastic fluid from rice leaves by the infiltration-centrifugation method.</a> Plant Cell Physiol. 53 (9), 1659-1668 (2012).</li><li>Rumyantseva, N. I., Valieva, A. I., Kostyukova, Y. A., Ageeva, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+leaf+plasticity+on+the+isolation+of+apoplastic+fluid+from+leaves+of+tartary+buckwheat+plants+grown+in+vivo+and+in+vitro.">The effect of leaf plasticity on the isolation of apoplastic fluid from leaves of tartary buckwheat plants grown in vivo and in vitro.</a> Plants. 12 (23), 4048 (2023).</li><li>Ekanayake, G., Gohmann, R., Mackey, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+quantitation+of+apoplast+hydration+in+Arabidopsis+leaves+reveals+water-soaking+activity+of+effectors+of+Pseudomonas+syringae+during+biotrophy.">A method for quantitation of apoplast hydration in Arabidopsis leaves reveals water-soaking activity of effectors of Pseudomonas syringae during biotrophy.</a> Sci Rep. 12 (1), 18363 (2022).</li><li>Preston, G. M., Fones, H. N., Mccraw, S., Rico, A., O'leary, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+infiltration-centrifugation+technique+for+extraction+of+apoplastic+fluid+from+plant+leaves+using+phaseolus+vulgaris+as+an+example.">The infiltration-centrifugation technique for extraction of apoplastic fluid from plant leaves using phaseolus vulgaris as an example.</a> J Vis Exp. (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. 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=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. J. <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>

Att använda växter för att producera terapeutiska proteiner har ökat snabbt de senaste åren 1,2,3,4,5,6. Proteinkodande gener klonas till expressionsvektorer och levereras till växtvävnader via agroinfiltration. Efter produktion av växtceller renas proteiner för nedströmsapplikationer. Vanligtvis renas rekombinanta ...

Numera studeras olika växtproducerade rekombinanta terapeutiska proteiner, inklusive antikroppar, vacciner, bioaktiva proteiner, enzymer och små polypeptider 1,2,3,4,5,6. Växter blir en alltmer använd plattform för att producera terapeutiska proteiner på grund av deras säkerhet, låga kostnad och förmåga att snabbt skala upp produktionen 7,8. Proteinuttryck och modifiering i växtsystem, tillsammans med proteinrening, är kritiska faktorer som bestämmer produktiviteten hos växtbioreaktorer 9,10. Att förbättra växternas produktivitet och erhålla målproteiner med hög renhet är centrala utmaningar som växtbaserade produktionssystem står inför11,12.
Den subcellulära lokaliseringen och modifieringen av rekombinanta proteiner beror på den specifika signalpeptid som kodas vid N-terminalen, som riktar proteinet till dess slutliga organelldestination under genuttryck. Rekombinanta proteiner är utformade för att lokaliseras till apoplast, endoplasmatiskt retikulum (ER), kloroplast, vakuol eller cytoplasma, baserat på deras unika egenskaper13. För antigener och antikroppar är utsöndrade proteiner att föredra. Före sekretion utvecklas proteiner genom ER och Golgi för korrekt veckning och posttranslationella modifieringar 14,15,16.
Efter produktion i växter måste rekombinanta proteiner renas från växtextrakt. Vanligtvis renas proteiner från totalt lösliga växtextrakt, som innehåller rikligt med intracellulära proteiner, felveckade och omodifierade produkter och cytokromer17. För att avlägsna föroreningar genomgår växtextrakt omfattande fraktionering, inklusive ribulos-1,5-bisfosfatkarboxylas/oxygenas (RuBisCO)-specifik affinitetskromatografi, fytatfällning, polyetylenglykolutfällning och justering av temperatur och pH18,19. Sådana steg för att avlägsna föroreningar är mödosamma och kan äventyra proteinkvaliteten.
Växtcellfacket bortom plasmamembranet definieras som apoplasten, som omfattar cellväggen, intercellulära utrymmen och xylem20. Den vattenhaltiga fasen kallas vanligtvis apoplast tvättvätska (AWF). AWF innehåller molekyler som utsöndras av celler för att reglera många biologiska framsteg som transport, cellväggsmetabolism och stressreaktioner21,22. Till skillnad från TSP är de molekylära beståndsdelarna i AWF mindre komplexa. Extraktion av rekombinanta proteiner från AWF kan undvika kontaminering av intracellulära proteiner, felveckade produkter och cytoplasmatiska rester. Kortfattat kommer extraktionslipid in i bladen genom klyvöppningarna under negativt tryck, och AWF samlas in genom mild centrifugering23. Även om isolering av AWF används i stor utsträckning för att studera de biologiska framstegen inom apoplast 24,25,26,27, har det inte utnyttjats i växtbaserade produktionssystem.
Att förbättra växternas produktivitet och erhålla målproteiner med hög renhet är centrala utmaningar för växtproduktionssystem28. Vanligtvis renas proteiner från totalt lösliga växtextrakt som innehåller stora mängder intracellulära proteiner, felveckade och omodifierade produkter och cytokromer29, vilket kräver stora kostnader för efterföljande rening30. Användningen av AWF-proteinextraktionsmetoder för extraktion av exogena rekombinanta proteiner som utsöndras i apoplast kan effektivt förbättra homogeniteten hos rekombinanta proteiner och minska kostnaden för proteinrening. Här använder vi en signalpeptid PR1a för att möjliggöra exogen proteinsekretion i apoplastutrymmet och introducerar en detaljerad metod för rekombinant proteinrening från AWF av 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  

Växter är ett nyutvecklat eukaryot uttryckssystem som utforskas för att producera terapeutiska proteiner. Rening av rekombinanta proteiner från växter är ett av de mest kritiska stegen i produktionsprocessen. Vanligtvis renades proteiner från totalt lösliga proteiner (TSP), och närvaron av diverse intracellulära proteiner och cytokromer utgör utmaningar för efterföljande proteinreningssteg. Dessutom utsöndras de flesta terapeutiska proteiner som antigener och antikroppar för att erhålla korrekt glykosylering, och närvaron av ofullständigt modifierade proteiner leder till inkonsekventa antigen- eller antikroppsstrukturer. Detta arbete introducerar en mer effektiv metod för att erhålla högrenade rekombinanta proteiner från det apoplastiska växtutrymmet. Det rekombinanta gröna fluorescerande proteinet (GFP) är konstruerat för att utsöndras i apoplasten av Nicotiana benthamiana och extraheras sedan med hjälp av en infiltrations-centrifugeringsmetod. GFP-His från den extraherade apoplasten renas sedan med nickelaffinitetskromatografi. Till skillnad från de traditionella metoderna från TSP, producerar rening från apoplasten högrenade rekombinanta proteiner. Detta innebär en viktig teknisk förbättring för växtproduktionssystemen.

Nowadays, various plant-produced recombinant therapeutic proteins are under study, including antibodies, vaccines, bioactive proteins, enzymes, and small polypeptides1,2,3,4,5,6. Plants are becoming an increasingly utilized platform for producing therapeutic proteins due to their safety, low cost, and ability to rapidly scale production7,8. Protein expression and modification in plant systems, along with protein purification, are critical factors determining the productivity of plant bioreactors9,10. Enhancing plant productivity and obtaining high-purity target proteins are core challenges facing plant-based production systems11,12.
The subcellular localization and modification of recombinant proteins depends on the specific signal peptide encoded at the N-terminus, which targets the protein to its final organelle destination during gene expression. Recombinant proteins are designed to localize to the apoplast, endoplasmic reticulum (ER), chloroplast, vacuole, or cytoplasm, based on their unique properties13. For antigens and antibodies, secreted proteins are preferred. Before secretion, proteins progress through ER and Golgi for correct folding and post-translational modifications14,15,16.
After production in plants, recombinant proteins must be purified from plant extracts. Typically, proteins are purified from total soluble plant extracts, which contain abundant intracellular proteins, misfolded and unmodified products, and cytochromes17. To remove impurities, plant extracts undergo extensive fractionation, including ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO)-specific affinity chromatography, phytate precipitation, polyethylene glycol precipitation, and adjustment of temperature and pH18,19. Such impurity-removal steps are laborious and can compromise protein quality.
The plant cell compartment beyond the plasma membrane is defined as the apoplast, encompassing the cell wall, intercellular spaces, and xylem20. The aqueous phase is commonly called apoplast washing fluid (AWF). AWF contains molecules secreted by cells to regulate numerous biological progresses like transport, cell wall metabolism, and stress responses21,22. In contrast to TSP, the molecular constituents of AWF are less complex. Extracting recombinant proteins from AWF may avoid contamination by intracellular proteins, misfolded products, and cytoplasmic remnants. Briefly, extraction lipid enters leaves through the stomata under negative pressure, and the AWF is collected by gentle centrifugation23. While isolation of AWF is widely employed to study the biological progress inside apoplast24,25,26,27, it has not been exploited in plant-based production systems.
Improving plant productivity and obtaining high purity target proteins are central challenges for plant production systems28. Typically, proteins are purified from total soluble plant extracts containing large amounts of intracellular proteins, misfolded and unmodified products, and cytochromes29, which require great costs for subsequent purification30. The use of AWF protein extraction methods for the extraction of exogenous recombinant proteins secreted into apoplast can effectively improve the homogeneity of recombinant proteins and reduce the cost of protein purification. Here, we use a signal peptide PR1a to enable exogenous protein secretion into the apoplast space and introduce a detailed method for recombinant protein purification from the AWF of N. benthamiana.

Författare i fokus: Optimera Apoplast-proteinextraktion för effektiv återvinning av rekombinanta proteiner från växtceller

Apoplast-extraktionsbaserad metod för att förbättra renheten hos växtproducerat rekombinant protein  

We investigated how to extract recombinant proteins from plant cells more efficiently. We answered the question of whether the apoplast protein extraction method is feasible when applied to the recombinant proteins. This extraction method extracts proteins secreted into the apoplast, so they have homogeneity.There is no need for cell disruption, which reduce the interference from intracellular proteins and the subsequent purification costs. We will focus on how to increase the secretion of recombinant proteins in the apoplast space to further improve the efficiency of the apoplast protein extraction method.

GFP-His är måltavla för utsöndring i det apoplastiska utrymmet av N. benthamiana GFP klonades in i pCAMBIA1300-plasmiden med en N-terminal PR1a-signalpeptid för sekretion och en C-terminal His-tagg för rening, vilket genererade p35s-PR1a-GFP-His (Figur 1A). Det rekombinanta proteinet uttrycktes tillfälligt i N. benthamiana och ett 30 kDa-band detekterades med western blot med användning av anti-HIS-antikroppar 3 dagar efter agroi...

Reningen av rekombinanta proteiner från växtsystem utmanas vanligtvis av växtproteiner. Detta arbete ger en metod för att effektivt extrahera och rena ett utsöndrat rekombinant protein från apoplasten av Nicotiana benthamiana.

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  

Plants are a newly developing eukaryotic expression system being explored to produce therapeutic proteins. Purification of recombinant proteins from plants is one of the most critical steps in the production process. Typically, proteins were purified from total soluble proteins (TSP), and the presence of miscellaneous intracellular proteins and cytochromes poses challenges for subsequent protein purification steps. Moreover, most therapeutic proteins like antigens and antibodies are secreted to obtain proper glycosylation, and the presence of incompletely modified proteins leads to inconsistent antigen or antibody structures. This work introduces a more effective method to obtain highly purified recombinant proteins from the plant apoplastic space. The recombinant Green fluorescent protein (GFP) is engineered to be secreted into the apoplast of Nicotiana benthamiana and is then extracted using an infiltration-centrifugation method. The GFP-His from the extracted apoplast is then purified by nickel affinity chromatography. In contrast to the traditional methods from TSP, purification from the apoplast produces highly purified recombinant proteins. This represents an important technological improvement for plant production systems.

The purification of recombinant proteins from plant systems is usually challenged by plant proteins. This work provides a method to effectively extract and purify a secreted recombinant protein from the apoplast of Nicotiana benthamiana.

Extraction and Purification of a Secreted Recombinant Protein from the Apoplast of Nicotiana benthamiana

To observe subcellular localization of GFP, after 48 hours of agroinfiltration of Nicotiana benthamiana, select a flat leaf and make a six-millimeter hole using a hole punch. Drip 30%sucrose solution onto a glass slide. Place the leaf sample with the back facing upwards, and cover it with a cover glass.Collect the intact, fresh N.benthamiana leaves expressing recombinant GFP at three days post-agroinfiltration, and weigh the leaves. Rinse the leaf blade with pre-cooled, sterilized, double-distilled water to remove debris. Submerge 20 grams with the abaxial side down in 500 milliliters of pre-cooled 100-millimolar phosphate buffer in a 1, 000 milliliter wide-mouth beaker.Cover the leaves with 100 mesh nylon, fine mesh yarn and a plastic plate. Connect the beaker to a vacuum pump. Apply 0.8 megapascal vacuum for one minute, then quickly restore to atmospheric pressure.Remove leaves from the buffer and gently blot dry with absorbent paper. Roll up the leaves and wrap them with mesh yarn. For collecting apoplastic washing fluid, or AWF, position rolled leaves tip-side up in 50-milliliter centrifuge tubes with mesh yarn fastened by the tube caps.Centrifuge the leaves at 500g for five minutes at four degrees Celsius. Remove the leaves and collect AWF using a pipette. Add Nickel Sepharose excel resin to a tube in a one-to-1, 000 ratio relative to the protein extract volume.Wash nickel resin three times with 10 times the resin volume of double-distilled water and phosphate buffer separately, in turn. Incubate protein extract with nickel resin for two hours to allow full binding. Then, centrifuge at 500g for five minutes at four degrees Celsius to remove unbound proteins in the supernatant.Wash three times with 20 times the resin volume of phosphate buffer. After the last wash, add 10 times the resin volume of 250 millimolar imidazole to elute GFPs. Centrifuge the mixture at 500g for five minutes at four degrees Celsius, and collect the supernatant.Western blot analysis confirmed the transient expression of 30 kilodalton recombinant protein GFPs in N.benthamiana. Fluorescence microscopy illustrated the presence of GFPs within the apoplast. AWF proteins extracted by vacuum infiltration centrifugation also contained GFPs.Out of the eight extraction solutions tested, phosphate buffer extracted the greatest amount of AWF and GFPs. Comparing phosphate buffer at varying pH revealed improved AWF and GFPs extraction at neutral to mildly alkaline conditions. Additionally, 100 millimolar phosphate buffer showed maximal efficacy for recovering AWF and GFPs.With a 100-millimolar phosphate buffer, 495 micrograms of total AWF proteins per gram of fresh leaf were recovered from the apoplast. The extracted AWF contained 10 micrograms of GFPs, which corresponded to approximately 18%of the GFPs in the total soluble protein. Following nickel affinity chromatography, GFPs'purity reached 84.3%from AWF extraction, substantially higher than the 44.9%purity achieved through total soluble protein extraction.