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

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. <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. 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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>

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 ...

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.

Biyoloji

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.