Name: comparison of tobacco host cell protein removal methods by blanching intact plants or by heat treatment of extracts
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We would like to acknowledge Dr. Thomas Rademacher, Alexander Boes and Veronique Bei&#223; for providing the transgenic tobacco seeds, and Ibrahim Al Amedi for cultivating the tobacco plants. The authors wish to thank Dr. Richard M. Twyman for editorial assistance as well as G&#252;ven Edg&#252; for providing the MSP1-19 reference. This work was funded in part by the European Research Council Advanced Grant ''Future-Pharma'', proposal number 269110, the Fraunhofer-Zukunftsstiftung (Fraunhofer Future Foundation) and Fraunhofer-Gesellschaft Internal Programs under Grant No. Attract 125-600164.

1. Tütün bitkileri yetiştirmek <ol><li> 0.1 [% ağırlık / hacim] gübre çözeltisi 1 I 1 L deiyonize su 2&#39;ye ve daha sonra her bir mineral yün blok yıkayın. Bir tütün her mineral yün bloğunda tohum ve tohum 16 yıkayarak olmadan gübre çözeltisi 0.25 L nazikçe yıkayın yerleştirin. </li><li> % 70 nispi nemde bir serada, 16 saat fotoperiyot 7 hafta içinde tütün bitkileri yetiştirmek (1 - m - 180 umol sn 2; λ = 400-700 nm) ve 25/22 ° C a?...

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Yukarıda açıklanan ısı yağış için üç yöntem etkili bir şekilde kromatografik saflaştırma adımı 16,17 önce tütün Onör puanı kaldırabilirsiniz. Bunlar, örneğin, ilk ürün saflığı, damlama 29 salgılanmış proteinler ile sınırlıdır hepsi rhizosecretion 30 ya da santrifüjlü ekstraksiyon 31,32 artırılmasını amaçlayan diğer metodlar tamamlar. hedef protein fazla 1 dakika boyunca ~ 60 ° C&#39;de en az çökelme sıcaklığ?...

Modern sağlık sistemleri giderek biofarmasötik proteinler 1 bağlıdır. Bitkilerde bu proteinlerin üretilmesi bilinen ekspresyon sistemleri 2-4 ile karşılaştırıldığında, düşük patojen yükü ve ölçeklenebilirlik avantajlıdır. yıkıcı çıkarma prosedürleri bulanıklıkları 5000 nefelometrik bulanıklık üniteleri (NTU) aşan ve konak hücre proteini (HCP) konsantrasyonları genellikle 95 aşan, yüksek parçacık yükü neden Ancak, bitkisel kökenli ilaçların aşağı işleme (DSP) zor olabilir % [m / m] 5,6. Ayrıntılı açıklama prosedürleri dağınık parçacıklar 7-9 kaldırmak için gerekli, ancak kromatografi cihazı CYp&#39;lerine 10,11 verimli çıkarılması için önceki bir adım varsa, ilk ürün kurtarma sırasında bağlama-ve-uzaklaştırma modunda çalışmaya daha pahalıdır vardır. Bu floccul kullanılarak hedef protein çökeltme yoluyla elde edilebilirkarıncalar 12 ya da düşük pH 13,14 yanı sıra HCp&#39;ler araya neden olarak. Ribuloz-1,5-bisfosfat karboksilaz / oksijenaz (RUBISCO), tütün (Nicotiana tabacum), yeşil bitkilerde en bol HCP, seçici çekiş polietilen glikol 15 eklenmesi ile geliştirilebilir, ancak bu, pahalı ve geniş ile uyumsuz olabilir ölçeğindeki üretim. Isıl işlem örneğin Vax8 protein sıtma aşısı adayları çözelti 16-18 kararlı kalırken, denatüre ve tütün CYp&#39;lerine fazla% 95 çökeltilmesi için gösterilmiştir. Sıcaklık kontrollü bir kap karıştırılır (ii) (i) beyazlatma, örneğin, sıcak sıvı içinde sağlam yaprak daldırma, ve (iii) bir ısı eşanjörü (: Üç farklı yaklaşımlar, tütün CYp&#39;lerine ısı kaynaklı çökelmesini elde etmek için kullanıldı Şekil 1) 16. sağlam yaprakları, CYp&#39;lerine hızlı ve verimli yağış elde etti ve aynı zamanda kolay gerçekleştirilen ağartmabüyütmek ve bitki biyokütlesi 19 yıkamak için bir başlangıç ​​aşamasını içerir mevcut büyük ölçekli üretim süreçleri ile uyumlu için. Bunun aksine, sıcaklık kontrollü DAMARLARIN bazı proseslerde kullanılabilir ve tank yüzey-hacim oranı, giderek azalır, çünkü bitkinin ısıl işlem 20 özler, ancak ölçeklenebilirlik ve enerji transferi hızı sınırlıdır için kullanılabilir ve süreç ölçeğinde uygun hale gelir. Bir ısı değiştirici de karıştırma kazanları ısıtıldı için teknik olarak iyi tanımlanmış bir alternatiftir ancak ısıtma ve soğutma ortamı, örn, buhar ve soğuk su bol miktarda bir kaynağı gibi ısı değiştirici geometrisine adapte edilmiş bir sıkı bir şekilde kontrol hacimsel akış hızı gerektirir medya özellikleri, örneğin., özgül ısı kapasitesi. Bu makalede, tüm üç yöntem, genel olarak, tütün CYp&#39;lerine ve bitki CYp&#39;lerine ısı kaynaklı çökeltme için nasıl kullanılabileceğini göstermektedir. kurulması ve EAC operasyonbir laboratuvar ortamında H yöntemi büyük ölçekli işlemler için uygunluklarını değerlendirmek için de kullanılabilir. büyük bir sorun süreci ölçekli üretim sırasında kullanılan cihazlar ve şartları andırır her işlem için yeterli ölçek aşağı modelleri ve çalışan koşullarını tespit etmektir. Burada sunulan veriler DsRed 16 sıtma aşısı adayı Vax8 floresan proteini ifade eden transgenik tütün bitkileri ile yapılan deneyler bakınız, ancak yöntem de başarılı N&#39;ye uygulanmış benthamiana bitkiler geçici diğer biofarmasötik proteinleri 21 dile getirdi. Bir tasarım-of-deneyler (DoE) işlem gelişimini kolaylaştırabilir 22 yaklaşım ve daha önce 8 açıklandığı gibi 23 de bu bağlamda yararlı olabilir flokülantlar. , Haşlama ısıtmalı kap ve eşanjör arasındaki temel fark bu beyazlaşma ot ise sürecin başlarında sağlam yapraklara uygulanan bironun bitki ekstreleri (Şekil 1) uygulanır. <img alt="Şekil 1" src="/files/ftp_upload/54343/54343fig1.jpg" /> Şekil 1:. Tütün HCP Isı Yağış Üç Farklı Yöntemlerle uygulanmasını gösteren Süreç Akış Şeması bitki materyali yıkanmış ve açıklama ve arıtma önce homojenize edilir. ağartma adımı (kırmızı) için donatım kolayca mevcut makinelerin eklenebilir. Buna karşılık, bir karıştırılan gemi (turuncu) ve özellikle eşanjör (mavi) kullanarak bir veya birden fazla ek aygıtlar ve boru gerektirir. <a href="https://www.jove.com/files/ftp_upload/54343/54343fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a>

<table border="0" cellpadding="0" cellspacing="0" width="1026">
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			<td>Software to operate the Zetasizer Nano ZS device</td>
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	</tbody>
</table>

comparison of tobacco host cell protein removal methods by blanching intact plants or by heat treatment of extracts

Bitkiler insanlar için yiyecek, yem ve hammadde sağlamak değil, aynı zamanda bu antikorların, aşı adayları ve enzimler gibi biyofarmasötik proteinler, ekonomik bir üretim sistemi olarak geliştirilmiştir değil sadece. Bu bitki biyokütlesinin saflaştırılması gerekir, ancak kromatografi aşamaları, bitki ekstrelerinde konakçı hücre proteinleri (HCp&#39;ler) yüksek konsantrasyonlarda girmesi engellenir. Bununla birlikte, çoğu HCp&#39;ler geri dönüşü olmayan hedef proteinin daha sonraki saflaştırmayı kolaylaştıran 60 ° C&#39;nin üzerindeki sıcaklıklarda bir araya getirir. Burada, üç yöntem sağlam yapraklar ya da özleri ya tütün CYp&#39;lerine ısı yağış elde etmek için sunulmuştur. bozulmamış yaprakların blanching kolayca mevcut süreçlere dahil edilebilir ancak sonraki filtrasyon adımlar üzerinde olumsuz bir etkiye sahip olabilir. tersi gibi proses ekipman tasarımı büyük değişiklikler ile de olsa alt işlemleri performansını artırmak, karıştırılan kap içinde, yaprak ekstreleri ısı çökeltilmesi için de geçerlidirhomojenleştirici geometrisi. Son olarak, bir ısı değiştirici kurulumu iyi ısı transfer koşulları açısından karakterize ve ölçek kolay, ama temizlik zor olabilir ve filtre kapasitesi üzerinde olumsuz bir etki olabilir edilir. Tasarım-of-deneyler yaklaşımı HCP kaldırma ve ürün kurtarma etkileyen en uygun proses parametrelerini belirlemek için kullanılabilir. Bu, diğer sentezleme platformlarda her yöntemi ve belirli bir saflaştırma stratejisi için en uygun yöntem tanımlanması uygulamayı kolaylaştırır.

Beyazlaşma tütün konak hücre proteinlerinin ısı yağış Bölüm 2&#39;de açıklanan beyazlatma işlemi başarılı bir şekilde, tütün 96 ±% 1 TSP azaltarak 70 ° C ile yapraklardan Onör puanı çökeltmek için kullanılabilir (n = 3) bu şekilde, Vax8 hedef proteinin% 51 kadar geri kazanılması artan ise kromatografik ayırma 16 önce% 0.1% 1.2 ila saflık. 64.1% 3.3 den% saflığı artan floresan proteini DsRed 83 ± 1% (n = 3) kurtarmak da müm...

Watch this Scientific Journal Video about Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts at JoVE.com

Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts

etkili bir tütün konakçı hücre proteinleri (HCp&#39;ler) arasında% 90&#39;dan daha fazla kaldırma çöktürme yöntemler sunulmuştur üç ısı başka saflaştırma aşamasından önce ayıklar. Bitki HCp&#39;ler tersinmez 60 ° C&#39;nin üzerindeki sıcaklıklarda bir araya getirir.

Bozulmamış Bitkiler Ağartma veya Ekstraktlarının Isıl İşlem tarafından Tütün Ana Hücre Proteini Temizleme Yöntemlerinin Karşılaştırılması

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical ...

Research

JoVE Journal

Biology

Preparation and Fractionation of Xenopus laevis Egg Extracts

Xenopus laevis Yumurta hazırlanması ve Fraksiyonu ayıklar.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

ELISA yöntemi ile yerli bozulmamış nükleozom Post-translasyonel modifikasyonlar Algılama

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

The MultiBac Protein Complex Production Platform at the EMBL

EMBL de MultiBac Protein Complex Üretim Platformu

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Tütün protoplastlarında ve Yapraklar Bimoleküler Floresan tamamlama tarafından Visualized protein-protein etkileşimleri

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Yetişkin Fare Kalvaryal Kemik İliği içinde Hematopoietik kök hücrelerin Vivo 4-Boyutlu Takip

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Fekal Glukokortikoid Analizi: Tek tırnaklılarda in Non-invaziv Adrenal İzleme

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

DamID seq: Adenin-metillenmiş DNA Fragments Yüksek Verimli Dizileme tarafından Protein-DNA Etkileşimleri genom Haritalama

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is ...

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts

Prosedür bitki özleri gelen Dağınık Parçacıkların çıkarması için Flokülantlar Verimliliği değerlendirin

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used ...

Comparison of Three Different Methods for Determining Cell Proliferation in Breast Cancer Cell Lines

Meme Kanseri Hücre Hatlarında Hücre Proliferasyonunu belirlenmesi için üç Farklı Yöntemlerle Karşılaştırılması

Measuring cell proliferation can be performed by a number of different methods, each with varying levels of sensitivity, reproducibility and compatibility ...

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

Xenopus Oositler: Mikroenjeksiyon için optimize Yöntemler, Foliküler Hücre Katmanlar çıkarılması ve Elektrofizyolojik Deneyler Hızlı Çözüm değişiklikler

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery ...