This work was supported by the COST action &#8216;Molecular pharming: Plants as a production platform for high-value proteins&#8217; FA0804. The Authors thank Dr Anatoli Giritch and Prof. Yuri Gleba for providing the MagnICON vectors for research purposes.

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The authors declare that there is no conflict of interests regarding the publication of this paper.

Bakteriyel hücreler, Bakulovirüs / böcek hücreleri ve bitkiler: Bu çalışmada üç farklı ortam, bir rekombinant insan proteininin sentezlenmesi için karşılaştırıldı. (- MagnICON ve pK7WG2 tabanlı - ve istikrarlı, yani geçici) bitki bazlı bir platform daha üç yaygın olarak kullanılan ifade teknolojilerini istismar tarafından araştırılmıştır. Bu deneyde, hGAD65mut için seçilen hedef protein önceden farklı sistemlerde 13 olarak ifade edildi ve üretim ve işlevselliği kolayca saptanabilir ve 14 ölçülebilir. HGAD65mut inklüzyon cisimcikleri oluşan çünkü bakteriyel hücreler, böylece zahmetli çözünürlüğünü gerektiren ve yerli yapısını elde etmek için yeniden katlama, hatta düşük sıcaklık koşullarında büyüyen, etkili bir üretim platformu değildi. Gerçekten de, kompleks rekombinant proteinlerin ifadesi için, bu platformun ana yetmezliği, son ürünün doğru konformasyon. Bakulovirüs / böcek hücresi platformu immünoreaktif yeniden birleştirici proteinin yüksek ifade ettiği, ancak bu ekspresyon sisteminin ana sınırlama böcek hücreleri büyütmek için gerekli kültür ortamı, yüksek maliyeti vardır. Bu hGAD65mut 1 g için toplam üretim maliyeti (gerekli böcek hücre kültür ortamı 9 L dikkate alınarak) bu üretim platformu 700 € ulaşabileceğini tahmin edilmiştir. Bu ifade platformunun bir diğer sınırlaması aseptik manipülasyon becerileri ile personel gerektirir böcek hücrelerinin steril ekimi, ihtiyaç vardır. Hücrelerin enfeksiyonu için kullanılır, viral stok hacimleri ve viral enfeksiyonun zamanlama iki kritik parametrelerin dikkatle Bu ekspresyon sistemi içinde kontrol edilmesi gereken etkin rekombinant proteini birikmesini sağlamak. Ayrıca, Sf9 böcek hücrelerinden elde edilen toplam çözünür protein çıkarımı için kullanılan deterjan, büyük ölçüde yeniden birleştirici proteinin çözünürlüğünü etkiler. Bitki bazlı sistemler en yararlı platfo vardırm hGAD65mut ifade: Bitki yapılan rekombinant protein immunoreaktif ve yaprak dokularında yüksek seviyelerde birikmiş oldu. Farklı bitki bazlı ekspresyon sistemleri karşılaştırıldığında, yüksek verimler stabil transforme edilmiş tütün bitkileri (Tablo 1), elde edildi, N&#39;ye göre tütün toplam biyomas dikkate halinde geçici ekspresyonu için kullanılan benthamiana, tütün tümüyle daha yüksek bir verimlilik belirgindir. Ancak, istikrarlı dönüştürülmüş tütün bitkisi-tabanlı platform ana sınırlama bizim çalışmamızda 20 ay sürdü sistemin set-up için gerekli zamandır. Gerçekten de, bir homozigot hattı yeniden birleştirici protein ekspresyonu homojenlik için seçilmelidir ve soyları yüksek T1 başlayarak, kendi kendine çapraz döngüleri tekrar gerektirebilir. Seçilen T1 transgenik özelliğin birden fazla kopyasını taşıyan zaman, özellikle, ıslah programı 3 yıla kadar sürebilir. Geçici sentezleme sistemleri sunulannedeniyle dönüşüm ve ifade ve anlatım platformu set-up arasında kısa aralıklarla hızlı yükseltme avantajı 4 gün gerekli. Ancak, bitki-temelli geçici sistemlerin bir sınırlama tarımsal infiltrasyon için özel yüksek-dereceli ekipmanları kullanılmadığı sürece kendi otomasyon laboratuvar ölçeğinde pek uygulanabilir olmasıdır. Bu nedenle, geçici tabanlı sistemler aracılığıyla hGAD65 büyük ölçekli üretim için uygun bir hesaplama burada gerçekleştirilemez. Öte yandan, biz T6 istikrarlı tütün hattı kullanarak rekombinant proteinin 1 g için toplam üretim maliyeti (60 tütün bitkileri yetiştirmek için toprağı dikkate alınarak) az 5 euro hesaplanabilir spekülasyon. (Bitki gelişim aşamasını büyümek ve Agrobacterium infiltrasyon durumu) birçok kritik parametreler dikkatle kontrol edilmelidir verimli agroinfiltration ve protein üretimini sağlamak, daha önce 15 rapor etmek. Bundan başka, her sentezleme için olması gereken belirli bir zaman süreci analizi denemerekombinant proteinin yüksek birikimini izin verir dpi seçmek için seslendirdi. Burada tartışılan örnek, geleneksel sistemlere göre bitki bazlı üretimin belirli bazı avantajlar vurgular proof-of-prensibi vaka çalışması, kabul edilebilir. Özellikle, homojen rekombinant proteini sentezleyen tütün transgenik çizgiler büyük miktarlarda gerekli olan yeniden birleştirici protein seri üretimi için değerli bir platform olarak kabul edilebilir.

Recombinant proteins are commercially mass-produced in heterologous expression systems with the aid of emerging biotechnology tools. Key factors that have to be considered when choosing the heterologous expression system include: protein quality, functionality, process speed, yield and cost.
In the recombinant protein field, the market for pharmaceuticals is expanding rapidly and, consequently, most biopharmaceuticals produced today are recombinant. Proteins can be expressed in cell cultures of bacteria, yeasts, molds, mammals, plants and insects, as well as in plant systems (either via stable- or transient-transformation) and transgenic animals; each expression system has its inherent advantages and limitations and for each target recombinant protein the optimal production system has to be carefully evaluated.
Plant-based platforms are arising as an important alternative to traditional fermenter-based systems for safe and cost-effective recombinant protein production. Although downstream processing costs are comparable to those of microbial and mammalian cells, the lower up-front investment required for commercial production in plants and the potential economy of scale, provided by cultivation over large areas, are key advantages.
We evaluated plants as bioreactors for the expression of the 65 kDa isoform of human glutamic acid decarboxylase (hGAD65), one of the major autoantigen in Type 1 autoimmune diabetes (T1D). hGAD65 is largely adopted as a marker, both for classifying and monitoring the progression of the disease and its role in T1D prevention is currently under investigation in clinical trials. If these trials are successful, the global demand for recombinant hGAD65 will increase dramatically.
Here, we focus on the expression of the enzymatically inactive counterpart of hGAD65, hGAD65mut, a mutant generated by substituting the lysine residue that binds the cofactor PLP (pyridoxal-5'-phosphate) with an arginine residue (K396R)1. 
hGAD65mut retains its immunogenicity and, in plant and insect cells, accumulates up to ten-fold higher than hGAD65, its wild-type counterpart. It was hypothesized that the enzymatic activity of hGAD65 interferes with plant cell metabolism to such an extent that it suppresses its own synthesis, whereas hGAD65mut, the enzymatically-inactive form, can be accumulated in plant cells to higher levels.
For the expression of hGAD65mut, the use of different technologies, widely used in plant biotechnology, was explored here and compared to traditional expression platforms (Escherichia coli and Baculovirus/insect cell-based).
In this work, the recombinant platforms developed for the expression of hGAD65mut comprising traditional and plant-based systems were reviewed and compared on the basis of process speed and yield, and of final product quality and functionality.

<table><tbody><tr><td>Yeast extract</td><td>Sigma</td><td>Y1333</td><td></td></tr><tr><td>Tryptone</td><td>Formedium</td><td>TRP03</td><td></td></tr><tr><td>Agar Bacteriological Grade</td><td>Applichem</td><td>A0949</td><td></td></tr><tr><td>Sf-900 II SFM medium</td><td>Gibco</td><td>10902-088</td><td></td></tr><tr><td>Grace&amp;rsquo;s Insect Medium, unsupplemented</td><td>Gibco</td><td>11595-030</td><td></td></tr><tr><td>Cellfectin II Reagent</td><td>Invitrogen</td><td>10362-100</td><td></td></tr><tr><td>MS medium including vitamins</td><td>Duchefa Biochemie</td><td>M0222</td><td></td></tr><tr><td>Sucrose</td><td>Duchefa Biochemie</td><td>S0809</td><td></td></tr><tr><td>Plant agar</td><td>Duchefa Biochemie</td><td>P1001</td><td></td></tr><tr><td>Ampicillin sodium</td><td>Duchefa Biochemie</td><td>A0104</td><td>Toxic</td></tr><tr><td>Gentamycin sulphate</td><td>Duchefa Biochemie</td><td>G0124</td><td>Toxic</td></tr><tr><td>Ganciclovir</td><td>Invitrogen</td><td>I2562-023</td><td></td></tr><tr><td>Carbenicillin disodium</td><td>Duchefa Biochemie</td><td>C0109</td><td>Toxic</td></tr><tr><td>Kanamycin sulfate</td><td>Sigma</td><td>K4000</td><td>Toxic</td></tr><tr><td>Rifampicin</td><td>Duchefa Biochemie</td><td>R0146</td><td>Toxic &amp;ndash; 25 mg/ml stock in DMSO</td></tr><tr><td>Streptomycin sulfate</td><td>Duchefa Biochemie</td><td>S0148</td><td>Toxic</td></tr><tr><td>Spectinomycin dihydrochloride</td><td>Duchefa Biochemie</td><td>S0188</td><td></td></tr><tr><td>IPTG (isopropil-&amp;beta;-D-1-tiogalattopiranoside)</td><td>Sigma</td><td>I5502</td><td>Toxic</td></tr><tr><td>MES hydrate</td><td>Sigma</td><td>M8250</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Biochemical</td><td>436994U</td><td></td></tr><tr><td>Acetosyringone</td><td>Sigma</td><td>D134406</td><td>Toxic &amp;ndash; 0.1 M stock in DMSO</td></tr><tr><td>Syringe (1 ml)</td><td>Terumo</td><td></td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fluka</td><td>63136</td><td></td></tr><tr><td>BAP (6-Benzylaminopurine)</td><td>Sigma</td><td>B3408</td><td>Toxic</td></tr><tr><td>NAA (Naphtalene acetic acid)</td><td>Duchefa Biochemie</td><td>N0903</td><td>Irritant</td></tr><tr><td>Cefotaxime</td><td>Mylan Generics</td><td></td><td></td></tr><tr><td>Trizma base</td><td>Sigma</td><td>T1503</td><td>Adjust pH with 1 N HCl to make Tris-HCl buffer</td></tr><tr><td>HCl</td><td>Sigma</td><td>H1758</td><td>Corrosive</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S3014</td><td>1 M stock</td></tr><tr><td>KCl</td><td>Sigma</td><td>P9541</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>S7907</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>P9791</td><td></td></tr><tr><td>PMSF (Phenylmethanesulfonylfluoride)</td><td>Sigma</td><td>P7626</td><td>Corrosive, toxic</td></tr><tr><td>Urea</td><td>Sigma</td><td>U5378</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Sigma</td><td>M3148</td><td>Toxic</td></tr><tr><td>Tween-20</td><td>Sigma</td><td>P5927</td><td></td></tr><tr><td>Hepes</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>DTT (Dithiothreitol)</td><td>Sigma</td><td>D0632</td><td>Toxic &amp;ndash; 1 M stock, store at -20 &amp;deg;C</td></tr><tr><td>CHAPS</td><td>Duchefa Biochemie</td><td>C1374</td><td>Toxic</td></tr><tr><td>Plant protease inhibitor cocktail</td><td>Sigma</td><td>P9599</td><td>Do not freeze/thaw too many times</td></tr><tr><td>SDS (Sodium dodecyl sulphate)</td><td>Sigma</td><td>L3771</td><td>Flammable, toxic, corrosive &amp;ndash; 10% stock</td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516</td><td></td></tr><tr><td>Brilliant Blue R-250</td><td>Sigma</td><td>B7920</td><td></td></tr><tr><td>Isopropanol</td><td>Sigma</td><td>24137</td><td>Flammable</td></tr><tr><td>Acetic acid</td><td>Sigma</td><td>27221</td><td>Corrosive</td></tr><tr><td>Anti-Glutamic acid decarboxylase 65/67</td><td>Sigma</td><td>G5163</td><td>Do not freeze/thaw too many times</td></tr><tr><td>Horseradish peroxidase (HRP)-conjugate anti-rabbit antibody</td><td>Sigma</td><td>A6154</td><td>Do not freeze/thaw too many times</td></tr><tr><td>Sf9 Cells</td><td>Life Technologies</td><td>11496</td><td></td></tr><tr><td>BL21 Competent &lt;em&gt;E. coli&lt;/em&gt;</td><td>New England Biolabs</td><td>C2530H</td><td></td></tr><tr><td>Protein A Sepharose</td><td>Sigma</td><td>P2545</td><td></td></tr><tr><td>Cell culture plates</td><td>Sigma</td><td>CLS3516</td><td></td></tr><tr><td>Radio Immuno Assay kit</td><td>Techno Genetics</td><td>12650805</td><td>Radioactive material</td></tr></tbody></table>

a comparative analysis of recombinant protein expression in different biofactories: bacteria, insect cells and plant systems

Bitki esaslı sistemler, yüksek kaliteli, biyolojik olarak aktif ürünler, esnek ve düşük maliyetli bir üretim için bunların iyi belgelenmiş potansiyel bir sonucu olarak, rekombinant proteinlerin üretimi için değerli bir platform olarak kabul edilir. Bu çalışmada, geçici ve stabil olarak bitki bazlı ekspresyon sistemleri, geleneksel fermentasyon cihazı dayalı hücre kültürlerinde (bakteriyel ve böcek) ve hedef insan yeniden birleştirici proteinin ifadesini karşılaştırıldı. Her platformda için, biz set-up, optimizasyon ve üretim sürecinin uzunluğu, nihai ürün kalitesi ve verimi açıklanan ve bizim seçtiğimiz hedef rekombinant protein için özel geçici üretim maliyetlerini, değerlendirildi. Genel olarak, sonuçlar bakteri bağlı erimez içerik korları dahilinde birikmesi, hedef proteinin üretimi için uygun olduğunu göstermektedir. Öte yandan, bitki bazlı sistemler, çok yönlü platformlar t&#39;ninşapka Bakulovirüs / böcek hücre sistemine daha düşük maliyetlerle seçilen proteinin üretimine izin verir. Özellikle, istikrarlı transgenik hatları nihai ürünün en yüksek verim ve geçici ifade bitkiler hızlı süreç geliştirme görüntülenen. Ancak, tüm rekombinant proteinleri bitki-temelli sistemlerde yararlanabilirler, ancak burada anlatıldığı gibi iyi üretim platformu, bir vaka-by-case yaklaşımı ile ampirik olarak tespit edilmelidir.

Farklı üretim sistemlerinde bir yeniden birleştirici hedef proteinin heterolog ekspresyonu için bir deney tasarımı burada tarif edilmektedir. ilk odak Her sistemde hedef proteinin ekspresyonu için uygun koşullar kurarak farklı düzlemlerin kurulum oldu. Hedef protein, hGAD65mut ekspresyonu, üçlü E. indüklenmiştir E. coli kültürleri. 37 ° C&#39;de ifade 3 saat sonra, bakteri hücreleri santrifüjleme ile toplandı ve sonikasyon ile parçalanır. Bir santrifüjleme kademesinden sonra, çözünür proteinler erimez içerik korları ayrılır ve ilk analizleri (veriler gösterilmemiştir) hGAD65mut çözünmez inklüzyon cisimcikleri içinde yaygın olarak birikmiş olduğu kanıtlanmıştır. Yeniden birleştirici protein çözünürlüğü, güçlü denatüre özellikleri, hGAD65mut imkansız uygun bir miktarının hale DEA analizi ile müdahale üre 6 M kullanımını gerektirdi. Birkaç stratejiler vartr, inklüzyon gövdelerinin oluşumunu sınırlayan düşük bir sıcaklıkta 11 mikrobiyal hücrelerin büyümesi ihtiva için rapor edilmiştir. Kültürler, 15 ° C ve 20 ° C&#39;de büyütüldü ve rekombinant protein ekspresyonu, aynı sıcaklıkta indüklenmiştir. Şekil 1A&#39;da gösterildiği gibi, hGAD65mut çözeltilmesi tekrar üre (şerit S2) gerektirir, her iki sıcaklıkta da üretilebilir. Bu durumda, bu deneyde düşük sıcaklıklarda çözülmeyen agrega şekillendirme hGAD65mut engellemez. HGAD65mut dizi (Baculo.G65mut) içeren baculovirüs vektörleri arasında yapışkan Sf9 hücre kültürlerinde ifade edilmiştir. V1 ve V2 yüksek titre stokları hazırlandı ve en iyi performans enfeksiyon koşulları 5-50 ul farklı viral stok hacimleri değerlendirilmesi kuruldu. Şekil 1 B &#39;de gösterildiği gibi, RIA ile değerlendirildiği üzere, uygun viral stok hacmi, kültür ortamının ml&#39;si başına rekombinant proteinin 11.8 ± 0.8 ug, sonuçta 25 ul olarak tanımlanmıştıranaliz (Tablo 1). Aşağıdaki sızma, agroinfected N. zaman ders analizi benthamiana yapraklarından iki geçici ifade sistemlerinde gerçekleştirildi. PK7WG2 tabanlı sistem için yaprak numuneleri aralığı 1-5 dpi günlük bir araya getirilmiş, toplam çözünür proteinleri (TSP) ile ekstre edildi ve TSP eşit miktarlarda western blot (Şekil 1C) ile analiz edilmiştir. Bu analiz, hedef rekombinant proteinin maksimum birikimi 2 dpi ulaşılır vurguladı. Bu nedenle, yaprak 2 dpi hasat edilmiştir ve protein ekstreleri 67.8 ug / g FLW (taze yaprak ağırlığı, Tablo 1) ortalama göstermektedir Rekombinant protein birikiminin ölçülmesi için, RIA ile analiz edilmiştir. Yeniden birleşen protein ifadesi seviyesi, bu sistem ile de 12 Pro, P19 ya da HC gibi bir transkripsiyon sonrası gen susturma (PTGS) içindeki bir bastırıcı ile yaprakların eş süzülmesiyle, iyileştirilebilir. Aynı zamanda ders algılama N. ile gerçekleştirildi benthamiana MagnICON vektörleri ile agroinfected yaprakları: enfekte yapraklar western blot ile analiz edilmiştir TSP 1-8 dpi ve eşit miktarlarda toplanmıştır. Bu analiz, RIA ile değerlendirildiği gibi, maksimum yeniden birleştirici proteinin birikmesi, 78.8 ug / g FLW (Tablo 1) yeniden birleştirici protein arasında bir ortalama birikimi, 4 dpi (Şekil 1D) elde edilir olduğunu gösterdi. Transgenik tütün bitkilerinde hGAD65mut sentezlenmesi, daha önce rekombinant protein seviyeleri bağımsız olarak dönüştürülen hatları arasında belirgin çeşitlilik gösteren, 12 bildirilmiştir. En iyi performans hGAD65mut T1 transgenik bitki öz-geçti ve elde edilen bitkiler (T2) tekrar iyi bir performans seçmek için analiz edildi. Bu prosedür yok kadar RIA tarafından her nesil performans denetimi, homojen bir üretim platformu geliştirmek için birkaç kuşak boyunca tekrarlandıDiğer bir gelişme (veriler gösterilmemiştir), elde edildi. Şekil 1 E&#39;deki T5 transjenik bitkilerin temsili bir Western blot yeniden birleştirici protein verimine homojenliği belirgin olduğu, bildirilmiştir. Şekil 1F&#39;de gösterildiği gibi, ortalama hGAD65mut verim seçim süreci boyunca 99.1 ug / g FLW (Tablo 1) ve bir seviyeye ulaşmıştır, T6, T2 yükselmiştir, tanım düzeyinde standart sapma azalmıştır. <img alt="Şekil 1," src="/files/ftp_upload/52459/52459fig1highres.jpg" /> Şekil 1:. Platform set-up her platformda hGAD65mut ifade için en iyi koşulları ayarlayın. (A), E. E. coli uyarılabilir ifade platformu. Bakteri hücreleri Coomassie ile lekelenmiş kontrol yükleme, 15 ° C ya da 20 ° C&#39;dir. Üst panel, hücre özütlerinde hGAD65mut batı lekesi (şerit başına 2 ug TSP). Alt panelde büyütülmüştür. n.c. = Negatif kontrol, kloramfenikol direnç geni içeren pDEST17 vektörü ile transforme edilen bakteri hücreleri; T: Toplam örnekleri; S1 Süpernatant, sonifikasyon ve santrifüjlemeden sonra çözünür; S2 ve P: Üst faz (1) ve Pelet (2) üre içeren bir tampon içinde çözülmüş örnek santrifüj sonrası. (B) Baculovirüs / böcek hücre platformu. sonraki viral stok hacmi test edilmiştir: 5, 25 ve 50 ul üst panel, hücre özütlerinde hGAD65mut batı benek (şerit başına 5 ug TSP) Alt panel, Coomassie ile lekelenmiş kontrol yükleme... nc negatif kontrol, dönüştürülmemiş böcek hücreleri özü =. N (C) Geçici sentezleme pK7WG2 vektörü kullanılarak benthamiana bitkileri. Numuneler, günlük 1-5 dpi (şerit 1-5). Üst panel, yaprak ekstrelerinde hGAD65mut batı leke (kulvar başına 2.5 ug TSP) toplandı. Alt paneli, Coomassie, büyük alt birim ile boyanmış denetimi yüklemedenRubisco&#39;nun açıktır. nc = Negatif kontrol, GFP işaretleyici geni taşıyan pK7WG2 vektörü ile infiltre bitkiler. (D), N. Geçici sentezleme MagnICON vektörler kullanılarak benthamiana bitkileri. Numuneler, günlük 1-8 dpi (şerit 1-8). Üst panel, yaprak ekstrelerinde hGAD65mut batı leke (kulvar başına 5 ug TSP) toplandı. Alt paneli, Coomassie ile boyanan denetimi yüklemeden Rubisco&#39;nun büyük alt birim belirgin olduğu . nc = negatif kontrol, sadece pICH20111 5&#39;-modül ve pICH14011 integraz-modülü ile sızmış bitkiler. Sayılar kDa molekül ağırlıklı işaretleyici göstermektedir. PC = pozitif kontrol, Bakulovirüs / böcek hücresi sistemi ile üretilmiş ticari hGAD65 15 ng. (E) N. Kararlı sentezleme Tabacum bitkiler. Yaprak numuneleri farklı T5 bitkiler (kulvarlar 1-11) elde edilmiştir. Üst panel, yaprak ekstrelerinde hGAD65mut batı leke (kulvar başına 5 ug TSP). Alt panel, yükleme kontrolü w lekelii Coomassie. nc negatif kontrol, vahşi tip bitkiler =. Sayılar kDa molekül ağırlıklı işaretleyici göstermektedir. PC = pozitif kontrol, Bakulovirüs / böcek hücresi sistemi ile üretilmiş ticari hGAD65 15 ng. (F), N. Kararlı sentezleme Tabacum bitkiler. Türetilen birçok nesiller boyunca hGAD65mut birikiminin Boxplot temsil DEA verilerinden hesaplanan ug / g flw olarak rapor hGAD65mut T1 transgenik tütün bitkisi performans en iyi şekilde. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52459/52459fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Sistem </td><td> [HGAD65mut] (ug / ml) </td><td> [HGAD65mut] </td></tr><tr><td> Bakülo / böcek </td><td> 117.5 ± 7.7 </td><td> 11.8 ± 0.8 ug / ml kültür ortamı </td></tr><tr><td> Transient </td><td> 22.6 ± 0.9 </td><td> 67.8 ± 2.7 ug / g FLW </td></tr><tr><td> MagnICON </td><td> 26.3 ± 5.9 </td><td> 78.8 ± 17.8 mg / g FLW </td></tr><tr><td> Elit T6 </td><td> 33.0 ± 3.8 </td><td> 99.1 ± 11.3 ng / g FLW </td></tr></tbody></table> Tablo 1: - fermenterler-tabanlı (Bakülo / Böcek) ve bitki-temelli (N. benthamiana pK7WG2- ve MagnICON ve N. tabacum elit T6) hGAD65mut farklı platformlarda hGAD65mut verim verir. İkinci sütun - protein ekstrelerindeki hGAD65mut konsantrasyonu (ug / ml). Üçüncü sütun - Bitki-tabanlı platformlar için ve fermenter-tabanlı platform için hücre kültür ortamında (ug / ml) taze yaprak ağırlığı (mg / g FLW) &#39;de hGAD65mut içeriği.

Watch this Scientific Journal Video about A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems at JoVE.com

Farklı Biyofabrikalarda Rekombinant Protein Ekspresyonunun Karşılaştırmalı Bir Analizi: Bakteriler, Böcek Hücreleri ve Bitki Sistemleri | Biyoloji | Video

A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems

In this study the expression of a target human recombinant protein in different production platforms was compared. We focused on traditional fermenter-based cultures and on plants, describing the set-up of each system and highlighting, on the basis of the reported results, the inherent limits and advantages for each platform.

Bakteriler, Böcek Hücreleri ve Bitki Sistemleri: Rekombinant Protein Farklı Biofactories İfade Karşılaştırmalı Analizi

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the ...

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23.4K Görüntüleme.  University of Verona, Verona, Italy. In this study the expression of a target human recombinant protein in different production platforms was compared. We focused on traditional fermenter-based cultures and on plants, describing the set-up of each system and highlighting, on the basis of the reported results, the inherent limits and advantages for each platform.

Video: A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell culture system. This system was developed to express foreign genes under the control of the baculoviral promoter in transient expression plasmids. Furthermore, this system can be applied to a functional assay of either the baculovirus itself or foreign proteins. The most widely and commercially available transient gene expression system is developed based on the immediate-early gene (IE) promoter of Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV). However, a low expression level of foreign genes in insect cells was observed. Therefore, a transient gene expression system was constructed for improving protein expression. In this system, recombinant plasmids were constructed to contain the target sequence under the control of the Drosophila heat shock 70 (Dhsp70) promoter. This protocol presents the application of this heat shock-based pDHsp/V5-His (V5 epitope with 6 histidine)/Spodoptera frugiperda cell (sf9 cell) system; this system is available not only for gene expression but also for evaluating the anti-apoptotic activity of candidate proteins in insect cells. Furthermore, this system can be either transfected with one recombinant plasmid or co-transfected two potentially functionally antagonistic recombinant plasmids in insect cells. The protocol demonstrates the efficiency of this system and provides a practical case of this technique.

Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay

This protocol describes a heat shock-induced protein expression system (pDHsp/V5-His/sf9 cell system), which can be used for either expressing foreign proteins or evaluating the anti-apoptotic activity of potential foreign proteins and their truncated amino acids in insect cells.

Geçici ifade, yabancı genlerin Protein fonksiyonel tahlil için böcek hücrelerindeki (sf9)

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell ...

Genetik

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The baculovirus-mediated insect cell expression system is one of the systems used to express recombinant eukaryotic secretory proteins with some post-translational modifications. The secretory proteins need to be routed through the secretory pathways for protein glycosylation, disulfide bonds formation, and other post-translational modifications. To improve the existing insect cell expression of secretory plant proteins, a baculovirus expression vector is modified by the addition of either a GP67 or a hemolin signal peptide sequence between the promoter and multiple-cloning sites. This newly designed modified vector system successfully produced a high yield of soluble recombinant secreted plant receptor proteins of Arabidopsis thaliana. Two of the expressed plant proteins, the extracellular domains of Arabidopsis TDR and PRK3 plasma membrane receptors, were crystallized for X-ray crystallographic studies. The modified vector system is an improved tool that can potentially be used for the expression of recombinant secretory proteins in the animal kingdom as well.

Here, we present the protocols for utilizing the insect cell and baculovirus protein expression system to produce large quantities of plant secreted proteins for protein crystallization. A baculovirus expression vector has been modified with either GP67 or insect hemolin signal peptide for plant protein secretion expression in insect cells.

Bitki proteinleri böcek hücrelerinde salgılanan Baculovirus ifade vektörel çizimler oluşturmak için değiştirme

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The ...

Biyokimya

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing&#160; a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

Developing biotinylatable fusion proteins has many potential applications in various fields of research. Recombinant protein engineering is a straight forward procedure that is cost-effective, providing high yields of custom-designed proteins.

Sinir doku rejenerasyonu için Çözünür ve Çözünmeyen Biyotinile Proteinlerin ifade, izolasyon ve saflaştırılması

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

Biyomühendislik

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

İnsan Hücrelerden salgılanan Proteinlerin Üretimi için Kullanışlı ve Genel Anlatım Platformu

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

Biyoloji

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

Rekombinant Proteinlerin Yüksek-düzey Geçici İfade için Bitkiler Verimli Agroinfiltration

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

Rekombinant Protein Yemler Kullanarak Phage Ekran ve Affinity seçimi için Protokol

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

Optimizasyon ve Kullanımı<em&gt; Agrobacterium</emIn&gt; aracılı Geçici Protein Üretimi<em&gt; Nicotiana</em

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells.

The expression of recombinant proteins by mammalian systems is becoming an attractive method for producing protein complexes for structural biology. Here we present a simple yet highly efficient expression system using suspension grown mammalian cells to purify protein complexes for structural studies.

Bir Roman ve Erişilebilir Yaklaşım: Yapısal HEK 293F Askı Hücreleri Biyoloji Rekombinant Protein

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two ...

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is the challenge of identifying suitable vaccine candidates. Malarial proteins expressed using prokaryotic and eukaryotic cell based expression systems are poorly glycosylated, generally insoluble and undergo improper folding leading to reduced immunogenicity. The wheat germ, rabbit reticulocyte lysate and Escherichia coli lysate cell free expression systems are currently used for expression of malarial proteins. However, the length of expression time and improper glycosylation of proteins still remains a challenge. We demonstrate expression of Plasmodium proteins in vitro using HeLa based cell free expression systems, termed &#8220;in vitro human cell free expression systems&#8221;. The 2 HeLa based cell free expression systems transcribe mRNA in 75 min and 3 &#181;l of transcribed mRNA is sufficient to translate proteins in 90 min. The 1-step expression system is a transcription and translation coupled expression system; the transcription and co-translation occurs in 3 hr. The process can also be extended for 6 hr by providing additional energy. In the 2-step expression system, mRNA is first transcribed and then added to the translation mix for protein expression. We describe how to express malaria proteins; a hydrophobic PF3D7_0114100 Maurer&#8217;s Cleft &#8211; 2 transmembrane (PfMC-2TM) protein, a hydrophilic PF3D7_0925900 protein and an armadillo repeats containing protein PF3D7_1361800, using the HeLa based cell free expression system. The proteins are expressed in micro volumes employing 2-step and 1-step expression strategies. An affinity purification method to purify 25 &#181;l of proteins expressed using the in vitro human cell free expression system is also described. Protein yield is determined by Bradford&#8217;s assay and the expressed and purified proteins can be confirmed by western blotting analysis. Expressed recombinant proteins can be used for immunizations, immunoassays and protein sequencing.

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

Expression of malarial proteins in cell based systems remains challenging. We demonstrate two step and one step IVT (in vitro translation) cell free expression systems for expressing malarial recombinant rhoptry proteins from HeLa cells. We use a Ni-resin affinity based purification system to purify the rhoptry proteins.

İfadesi için HeLa Tabanlı Hücre Özgür İfade Sistemleri<em&gt; Plasmodium</em&gt; Rhoptry Proteinler

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

Bakteriler, Böcek Hücreleri ve Bitki Sistemleri: Rekombinant Protein Farklı Biofactories İfade Karşılaştırmalı Analizi

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