Bu çalışma, Gençlik İnovasyonu Teşvik Derneği CAS (2021084) ve Çin Bilimler Akademisi Mikrobiyoloji Enstitüsü Üç Yıllık Eylem Planının Anahtar Dağıtım Projesi Destek Fonu tarafından desteklenmektedir.

1. N. benthamiana bitkilerinin hazırlanması
<ol><li>Fide bloklarını bir tepsiye koyun, 1 L su ekleyin ve tamamen dolana kadar yaklaşık 2 saat bekletin.</li>
	<li>Fide bloklarının üzerine eşit şekilde yabani tip N. benthamiana tohumlarını serpin, bir kapakla örtün ve 1 hafta boyunca çimleyin. N. benthamiana'yı 18 saat fotoperiyodu, 24 °C, %45 bağıl nemi olan bir serada yetiştirin ve her 2 haftada bir gübreleyin.</li>
	<l...

N. benthamiana Yapraklarından GFP-His'in Verimli Ekstraksiyonu ve Saflaştırılması

<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>

Yazarlar herhangi bir çıkar çatışması beyan etmemektedir.

Terapötik proteinler üretmek için bitkilerin kullanılması son yıllarda hızla genişledi 1,2,3,4,5,6. Protein kodlayan genler, ekspresyon vektörlerine klonlanır ve agroinfiltrasyon yoluyla bitki dokularına verilir. Bitki hücreleri tarafından üretildikten sonra, proteinler sonraki uygulamalar için saflaştırılı...

Günümüzde, antikorlar, aşılar, biyoaktif proteinler, enzimler ve küçük polipeptitler 1,2,3,4,5,6 dahil olmak üzere bitki tarafından üretilen çeşitli rekombinant terapötik proteinler incelenmektedir. Bitkiler, güvenlikleri, düşük maliyetleri ve üretimi hızlı bir şekilde ölçeklendirme yetenekleri nedeniyle terapötik proteinler üretmek için giderek daha fazla kullanılan bir platform haline gelmektedir 7,8. Bitki sistemlerinde protein ekspresyonu ve modifikasyonu, protein saflaştırması ile birlikte, bitki biyoreaktörlerininverimliliğini belirleyen kritik faktörlerdir 9,10. Bitki verimliliğini artırmak ve yüksek saflıkta hedef proteinler elde etmek, bitki bazlı üretim sistemlerinin karşılaştığı temel zorluklardır11,12.
Rekombinant proteinlerin hücre altı lokalizasyonu ve modifikasyonu, gen ekspresyonu sırasında proteini nihai organel hedefine hedefleyen N-terminalinde kodlanan spesifik sinyal peptidine bağlıdır. Rekombinant proteinler, benzersiz özelliklerine bağlı olarak apoplast, endoplazmik retikulum (ER), kloroplast, vakuol veya sitoplazmaya lokalize olacak şekilde tasarlanmıştır13. Antijenler ve antikorlar için salgılanan proteinler tercih edilir. Sekresyondan önce, proteinler doğru katlanma ve translasyon sonrası modifikasyonlar için ER ve Golgi boyunca ilerler 14,15,16.
Bitkilerde üretimden sonra, rekombinant proteinler bitki özlerinden saflaştırılmalıdır. Tipik olarak proteinler, bol miktarda hücre içi proteinler, yanlış katlanmış ve modifiye edilmemiş ürünler ve sitokromlar17 içeren toplam çözünür bitki özlerinden saflaştırılır. Safsızlıkları gidermek için bitki özleri, ribuloz-1,5-bifosfat karboksilaz/oksijenaz (RuBisCO) spesifik afinite kromatografisi, fitat çökeltme, polietilen glikol çökeltme ve sıcaklık ve pH 18,19'un ayarlanması dahil olmak üzere kapsamlı fraksiyonlamaya tabi tutulur. Bu tür safsızlık giderme adımları zahmetlidir ve protein kalitesini tehlikeye atabilir.
Plazma zarının ötesindeki bitki hücresi bölmesi, hücre duvarını, hücreler arası boşlukları ve ksilem20'yi kapsayan aplast olarak tanımlanır. Sulu faz genellikle apoplast yıkama sıvısı (AWF) olarak adlandırılır. AWF, taşıma, hücre duvarı metabolizması ve stres tepkileri gibi çok sayıda biyolojik ilerlemeyi düzenlemek için hücreler tarafından salgılanan molekülleri içerir21,22. TSP'nin aksine, AWF'nin moleküler bileşenleri daha az karmaşıktır. AWF'den rekombinant proteinlerin çıkarılması, hücre içi proteinler, yanlış katlanmış ürünler ve sitoplazmik kalıntılar tarafından kontaminasyonu önleyebilir. Kısaca, ekstraksiyon lipidi yapraklara negatif basınç altında stomalardan girer ve AWF nazik santrifüjleme23 ile toplanır. AWF'nin izolasyonu, apoplast24,25,26,27 içindeki biyolojik ilerlemeyi incelemek için yaygın olarak kullanılırken, bitki bazlı üretim sistemlerinde kullanılmamıştır.
Bitki verimliliğinin artırılması ve yüksek saflıkta hedef proteinlerin elde edilmesi, bitki üretim sistemleri için temel zorluklardır28. Tipik olarak, proteinler, büyük miktarlarda hücre içi proteinler, yanlış katlanmış ve modifiye edilmemiş ürünler ve sonraki saflaştırma için büyük maliyetler gerektirensitokromlar 29 içeren toplam çözünür bitki özlerindensaflaştırılır 30. Apoplast içine salgılanan eksojen rekombinant proteinlerin ekstraksiyonu için AWF protein ekstraksiyon yöntemlerinin kullanılması, rekombinant proteinlerin homojenliğini etkili bir şekilde artırabilir ve protein saflaştırma maliyetini azaltabilir. Burada, apoplast boşluğuna eksojen protein salgılanmasını sağlamak için bir sinyal peptidi PR1a kullanıyoruz ve N. benthamiana'nın AWF'sinden rekombinant protein saflaştırması için ayrıntılı bir yöntem sunuyoruz.

<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  

Bitkiler, terapötik proteinler üretmek için araştırılan yeni gelişen bir ökaryotik ekspresyon sistemidir. Bitkilerden rekombinant proteinlerin saflaştırılması, üretim sürecindeki en kritik adımlardan biridir. Tipik olarak, proteinler toplam çözünür proteinlerden (TSP) saflaştırıldı ve çeşitli hücre içi proteinlerin ve sitokromların varlığı, sonraki protein saflaştırma adımları için zorluklar doğurur. Ayrıca, antijenler ve antikorlar gibi terapötik proteinlerin çoğu, uygun glikozilasyon elde etmek için salgılanır ve tam olarak değiştirilmemiş proteinlerin varlığı, tutarsız antijen veya antikor yapılarına yol açar. Bu çalışma, bitki apoplastik alanından yüksek oranda saflaştırılmış rekombinant proteinler elde etmek için daha etkili bir yöntem sunmaktadır. Rekombinant Yeşil floresan proteini (GFP), Nicotiana benthamiana'nın apoplastına salgılanmak üzere tasarlanmıştır ve daha sonra bir infiltrasyon-santrifüjleme yöntemi kullanılarak ekstrakte edilir. Ekstrakte edilen apoplasttan elde edilen GFP-His daha sonra nikel afinite kromatografisi ile saflaştırılır. TSP'nin geleneksel yöntemlerinin aksine, apoplasttan saflaştırma, yüksek oranda saflaştırılmış rekombinant proteinler üretir. Bu, bitki üretim sistemleri için önemli bir teknolojik gelişmeyi temsil eder.

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.

Yazar Spotlight: Bitki Hücrelerinden Rekombinant Proteinlerin Verimli Bir Şekilde Geri Kazanılması için Apoplast Protein Ekstraksiyonunun Optimize Edilmesi

Bitki Tarafından Üretilen Rekombinant Proteinin Saflığını Artırmak için Apoplast-Ekstraksiyon Bazlı Yöntem  

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, N. benthamiana'nın apoplastik alanına salgılanması hedefleniyor GFP, sekresyon için bir N-terminal PR1a sinyal peptidi ve saflaştırma için bir C-terminal His-tag ile pCAMBIA1300 plazmidine klonlandı ve p35s-PR1a-GFP-His üretildi (Şekil 1A). Rekombinant protein N . benthamiana'da geçici olarak eksprese edildi ve agroinfiltrasyondan 3 gün sonra anti-His antikoru kullanılarak western blot ile 30 kDa bandı tespit edil...

Bitki sistemlerinden rekombinant proteinlerin saflaştırılması genellikle bitki proteinleri tarafından sorgulanır. Bu çalışma, Nicotiana benthamiana'nın apoplastından salgılanan bir rekombinant proteini etkili bir şekilde ekstrakte etmek ve saflaştırmak için bir yöntem sağlar.

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

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Research

JoVE Journal

Biology

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

625 Views.  Shandong First Medical University. 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 secretio...