Name: automated hydrophobic interaction chromatography column selection for use in protein purification
Uploaded: 2011-09-21T13:00:00.000+00:00
Duration: 10 min 21 s
Description: Watch this Scientific Journal Video about Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification at JoVE.com

Bu çalışma Sağlık hibe GM086822 ve Ulusal Bilim Vakfı başlıca araştırma enstrümantasyon hibe DBI-0.960.313 Ulusal Enstitüsü tarafından finanse edildi. Yazarlar Dr teşekkür etmek istiyorum. Jon Miyake ve Donna Hardy (Bio-Rad) ve teknik uzmanlık için Jennifer Loertscher (Seattle). Kromatografisi reaktifler seçin ve tamamlayıcı enstrümantasyon cömertçe Bio-Rad tarafından sağlanmıştır.

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophobic+interaction+chromatography+of+proteins.+IV.+Protein+adsorption+capacity+and+transport+in+preparative+mode.">Hydrophobic interaction chromatography of proteins. IV. Protein adsorption capacity and transport in preparative mode.</a> J. Chromatogr. A. 1218, 427-440 (2011).</li><li>Huddleston, J. 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C., Banh, C., Brossay, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L.A+Protocol+for+the+Production+of+KLRG1+Tetramer.">L.A Protocol for the Production of KLRG1 Tetramer.</a> J. Vis. Exp. (35), e1701-e1701 (2010).</li><li>Murphy, P. J. M., Morishima, Y., Kovacs, J. J., Yao, T. P., Pratt, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+dynamics+of+hsp90+action+on+the+glucocorticoid+receptor+by+acetylation/deacetylation+of+the+chaperone.">Regulation of the dynamics of hsp90 action on the glucocorticoid receptor by acetylation/deacetylation of the chaperone.</a> J. Biol. Chem. 280, 33792-33799 (2005).</li><li>Zeytuni, N., Zarivach, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+the+M.+magneticum+strain+AMB-1+Magnetosome+Associated+Protein+MamA&#916;41.">Purification of the M. magneticum strain AMB-1 Magnetosome Associated Protein MamA&#916;41.</a> J. Vis. Exp. (37), 10-3791 (2010).</li><li>Kong, Y., Li, X., Bai, G., Ma, G., Su, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automatic+system+for+multidimensional+integrated+protein+chromatography.">An automatic system for multidimensional integrated protein chromatography.</a> J. Chromatogr. A. 1217, 6898-6904 (2010).</li><li>Camper, D. V., Viola, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fully+automated+protein+purification.">Fully automated protein purification.</a> Anal. Biochem. 393, 176-181 (2009).</li><li>Hammon, J., Palanivelu, D. V., Chen, J., Patel, C., Minor, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+green+fluorescent+protein+screen+for+identification+of+well-expressed+membrane+proteins+from+a+cohort+of+extremophilic+organisms.">A green fluorescent protein screen for identification of well-expressed membrane proteins from a cohort of extremophilic organisms.</a> Protein Sci. 18, 121-133 (2009).</li><li>Wu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+green+and+red+fluorescent+proteins+to+teach+protein+expression,+purification,+and+crystallization.">Using green and red fluorescent proteins to teach protein expression, purification, and crystallization.</a> Biochem. Mol. Biol. Educ. 36, 43-54 (2008).</li><li>Queiroz, J. A., Tomaz, C. T., Cabral, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophobic+interaction+chromatography+of+proteins.">Hydrophobic interaction chromatography of proteins.</a> J. Biotechnol. 87, 143-159 (2001).</li><li>McCue, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theory+and+use+of+hydrophobic+interaction+chromatography+in+protein+purification+applications.">Theory and use of hydrophobic interaction chromatography in protein purification applications.</a> Methods Enzymol. 463, 405-414 (2009).</li></ol>

Kromatografisi reaktifler seçin ve tamamlayıcı enstrümantasyon Bio-Rad tarafından sağlanmıştır.

Sıvı kromatografi teknikleri 7 biyokimyasal, immünolojik 6 yürütülmesi için gerekli olan yüksek derecede saflaştırılmış protein hazırlanması, ve yapısal 8 çalışmalar için çok değerli olduğu kanıtlanmıştır. HIC saflaştırma yöntemleri çoğunlukla tercih edilen ortamın ampirik belirlenmesi gerekir, ve ligand yapısı, ligand yoğunluğu, ve matris boncuk özellikleri her kromatografik sonuçlar 2, 3 etkisi gösterilmiştir. Izcilik Otomatik sütun sonraki optimizasyonu ve protein saflaştırma 4 için bir HIC orta seçilmesi için etkili bir yaklaşımdır. Sunulan otomatik sütun izcilik yöntemi kolaylıkla çeşitli HIC protein saflaştırma stratejileri adapte edilebilir. Tuz konsantrasyonu, tuz seçimi, tuz degrade ve pH değişiklikler ilave arıtma hastalıkları iyileştirebilir ve bu temel parametreleri değişen etkileri daha önce 13,14 gözden geçirilmiştir. Kısmen saflaştırılmış hedef prote içeren HIC eluatdaha da bu tür iyon değişimi kromatografisi (IEX) veya jel filtrasyonu / boyut harici kromatografi 9,10 olarak tamamlayıcı bir kromatografik tekniği kullanılarak saflaştırılabilir. Çünkü eşsiz ışık absorbans ve emisyon karakteristikleri, GFP ile elüsyon profiline-line ve post-çalıştırma yaklaşımlar kullanılarak belirlenebilir. Bu amaçla, bu protokol daha da alakasız bir hedef protein GFP &quot;tagged&quot; olduğu, rekombinant GFP-füzyon proteinlerinin saflaştırılması için adapte edilebilir. GFP-etiketli hedef proteinin UV ışığı 11 ve yukarıda açıklanan HIC saflaştırma stratejisini içeren çeşitli kromatografik yaklaşımlar kullanılarak saflaştırıldı ile tespit edilebilir. GFP arıtma aynı zamanda modern protein bilimi teknikleri 12 öğretimi için biyokimya laboratuvarlarında pedagojik bir elyaf haline gelmiştir. Temel HIC protein saflaştırılması bir ölçüde daha az sağlam kromatografisi sy kullanarak yürütülebilirkenKök, burada sunulan enstrümantasyon olumlu sonuçlar elde yardımcı olmak için farklı bir takım avantajları vardır. Son derece otomatik bir sistem kullanmanın önemli yararları çalıştırmak çalıştırılmaya koşulu tekrarlanabilirlik, zaman tasarrufu da geliştirilmiş ve sistem 10 içine verilmek üzere hava için fırsatlar azalma sayılabilir. Sistem arttıran kolaylaştırılır Sistemi kontrollü tampon karıştırma, tampon hazırlanması ve deneysel tekrarlanabilirlik artan tutarlılık sağlar. Tüm izcilik için dinamik örnek döngü içine yeterince örnek yük Çizim çalışan başka her sütun eklenmeden örnek uyum sağlar ve kesinti veya yeniden manuel olarak sıralı çalışmaları için izin verir. Sütun üzerine örnek loop kontrollü numune enjeksiyonu el ile örnek yükleme ile oluşabilir değişkenliği azalır. Sütun seçimi valf bir çift sistemi replumb gerek kalmadan, ardışık Örnek ishal, farklı bir HIC kolonu kullanılarak her biri için olanak sağlar. Mul Sahne<html> eşsiz bir spektrofotometrik profil ile bir protein test edilmesinden sonra ti-dalgaboyu analizi özellikle faydalı olmaktadır. GFP ek olarak, sitokromlar, flavoproteins, ve diğer heme ihtiva eden proteinler Bu tekniğin yararlanabilir. İletkenlik ve pH izleme cihazları gerçek zamanlı deneysel koşullar doğrulanması için izin verir. Bölünmüş fraksiyonu eluat toplama geliştirilmiş fraksiyonu bir şekilde kullanılmasını sağlar ve minimal bir örnek hacmi (örneğin ELISA, SDS-PAGE, western blot ve Experion mikroakışkan elektroforezi) gerektiren post-run analiz yöntemleri için kolay aktarım sağlar. Ikinci fraksiyon toplayıcı çevrimdışı kullanma kılavuzu senkronizasyon gerektirir iken, fraksiyon toplayıcı seçimi maksimum esneklik sağlar. HIC izcilik sütun ve protein saflaştırma için böyle sağlam bir kromatografi sistemi kullanan için en önemli sakıncaları başlangıç ​​zamanı ve aracı satın alma ve operatör eğitimi ile ilgili bütçe harcamaları içerir. t &quot;&gt; Burada sunulan protokolü Bio-Rad Çift Geçişli kromatografi sistemi kullanır;. Ancak, bu tür GE Healthcare gelen Akta Avant diğer üreticilerin, eşit derecede sağlam enstrümantasyon da olabilir kullanıldığı ve eşdeğer sonuçlar üretmektedir bile karşılaştırılabilir kromatografi sistemi bir arınma prosedürü veya cihaz satın başlamadan önce düşünülmelidir benzersiz özellikleri (örneğin yöntemi programlama, bileşen adlandırma, operatör tercihi ve ölçeklenebilirlik sınırlamalar) var.

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> <a href="http://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=223301" target="_blank">Biyolojik Çift Geçişli Pathfinder 20 Sistemi</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td> <a href="http://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=223301" target="_blank">7602257</a> </td><td> Sistemi arttırıcı, mikser, F10 iş istasyonu, AVR7-3 valf, QuadTec UV / akış hücresi, BioFrac fraksiyon toplayıcı, biyolojik yazılımı ile Vis dedektör ve başlangıç ​​kiti içerir </td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=760-0408&vertical=LSR&parentCategoryGUID=22c495f1-36ff-44a3-9140-ee7e54d0b1ba" target="_blank">AVR9-8 akışı seçin valf</a><html></td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=760-0408&vertical=LSR&parentCategoryGUID=22c495f1-36ff-44a3-9140-ee7e54d0b1ba" target="_blank">7600408</a> </td><td> Biyolojik Çift Geçişli sistemi ile kullanım için yüksek basınç vana, 9-port, 8-konumunda, 3500 psi (233 bar) sınırı, </td></tr><tr><td> Yönlendirici vana SV3T-2 </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td> 7600410 </td><td> Biyolojik Çift Geçişli sistemi ile kullanım için selenoid vana, 3 yollu, 2 konumlu, 30 psi (2 bar) sınırı, </td></tr><tr><td> Akış ayrıştırıcı vana </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td></td><td></td></tr><tr><td>Tical = LSR &amp; parentCategoryGUID = 22c495f1-36ff-44a3-9140-ee7e54d0b1ba &quot;target =&quot; _blank &quot;&gt; DynaLoop 25 Kiti </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=750-0451&vertical=LSR&parentCategoryGUID=22c495f1-36ff-44a3-9140-ee7e54d0b1ba" target="_blank">7500451</a> </td><td> Örnek döngü kiti Sürme-piston, döngü sürgülü 25 ml DynaLoop, DynaLoop parça kiti (# 750-0450) içerir </td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/LSR/PDP/ec82e7b4-919e-428b-ac03-9adf1f50a48e/BioFractrade_Fraction_Collector" target="_blank">BioFrac Kesir Collector</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/LSR/PDP/ec82e7b4-919e-428b-ac03-9adf1f50a48e/BioFractrade_Fraction_Collector" target="_blank">7410002</a> </td><td> İki fraksiyon col<html> lectors kullanılan </td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=741-0088&vertical=LSR&parentCategoryGUID=ec82e7b4-919e-428b-ac03-9adf1f50a48e" target="_blank">BioFrac mikroplak damla kafa adaptörü</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=741-0088&vertical=LSR&parentCategoryGUID=ec82e7b4-919e-428b-ac03-9adf1f50a48e" target="_blank">7410088</a> </td><td></td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&vertical=LSR&country=US&lang=en&productID=741-0017" target="_blank">BioFrac mikrotitre plakası adaptörü</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td>&amp; Country = ABD &amp; lang = tr &amp; ProductID = 741-0017 &quot;target =&quot; _blank &quot;&gt; 7410017 </td><td></td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=750-0202&vertical=LSR&parentCategoryGUID=c140016b-1958-4a91-be4c-8645a4c8204f" target="_blank">UV Optik Modülü</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=750-0202&vertical=LSR&parentCategoryGUID=c140016b-1958-4a91-be4c-8645a4c8204f" target="_blank">7500202</a> </td><td></td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&isFromSearch=true&productID=760-1331&vertical=LSR&parentCategoryGUID=c4095dc1-9260-4431-b066-823b050048c8" target="_blank">Halojen lamba</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td> &lt;<html> Bir target = &quot;_blank&quot;&gt; 7601331 </td><td></td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=220901" target="_blank">Econo Gradient Pompası</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=220901" target="_blank">7319001</a> </td><td> düşük basınçlı protein saflaştırılması için gradient pompa, boru ve bağlantı parçaları kiti içerir </td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=224301" target="_blank">Experion Sistemi</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td>bio-rad.com/prd/en/US/adirect/biorad? cmd = BRCatgProductDetail &amp; ProductID = 224301 &quot;target =&quot; _blank &quot;&gt; 7.007.001 </td><td></td></tr><tr><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=224101" target="_blank">Experion Pro260 Analiz Kiti</a> </td><td> <a href="http://www.bio-rad.com/" target="_blank">Bio-Rad</a> </td><td> <a href="https://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=224101" target="_blank">7007102</a> </td><td></td></tr><tr><td> HiTrap HIC sütun seçimi kiti </td><td> GE Healthcare </td><td> 28-4110-07 </td><td> Izcilik için HIC medyanın 7 x 1 ml kullanıma hazır sütunlar içerir </td></tr><tr><td> GE Healthcare-to-Bio-Rad sütun parçaları </td><td> GE Healthcare </td><td> 18111251 ve 18111257 </td><td></td></tr></tbody></table>

automated hydrophobic interaction chromatography column selection for use in protein purification

Temizleyici proteinleri (örneğin, jel filtrasyonu, afinite, ve iyon değişimi) için diğer kromatografik metodlar aksine, hidrofobik etkileşim kromatografısi (HIC) yaygın olarak en uygun kromatografik orta seçmek için deneysel tayini (tarama ya da &quot;izcilik&quot; olarak anılacaktır) gerektirir Verilen bir proteinin saflaştırılması için 1. Burada anlatılan yöntem proteinin saflaştırılması kullanılmak üzere en uygun HIC ortam için izcilik için otomatik bir yaklaşımı tarif eder. HIC hidrofobiklik farklılıklara bağlı olarak bir ham lizat proteinleri ve diğer biyomoleküllerin ayırır. Afinite kromatografisi (AC) ve iyon değişimi kromatografisi (IEX) benzer şekilde, bu HIC kromatografi işlemi boyunca ilerlerken ilgili proteini konsantre yeteneğine sahiptir. Iyi HIC tarafından arıtma için uygundur Proteinler hidrofobik yüzey bölgeler ve 2 M cephane aşan konsantrasyonlarda tuz maruz dayanabilmesi olanlar dahilNeodinyum sülfat ((NH4) 2 SO4). HIC genellikle bir yakınlık etiketi eksik proteinler için bir arınma yöntemi olarak seçilmiş, ve AC için böylece uygun ve IEX yeterli arıtma sağlamak için başarısız olduğunda edilir. Proteininin yüzeyde hidrofobik yarımlar geçici bir inert, hareketsiz matris şekilde bağlanmış bir polar olmayan ligant bağlama. Protein ve ligand arasındaki etkileşimi protein-ligand etkileşimi güçlendirmek ve hareketsiz protein (yani sütun içindeki bağlı) 2 yaparak yüksek iyonik konsantrasyonları, kromatografi sütunu akan tampon tuz konsantrasyonu son derece bağımlıdırlar. Tuz konsantrasyonu düşüş olarak, protein-ligand etkileşimi dağıtır, protein tekrar sütundan mobil ve elutes hale gelir. Çeşitli HIC ortam önceden paketlenmiş kolon, ticari olarak mevcuttur, birkaç hidrofobik ligandları (örn. oktil S-butil, butil, ve fenil) her birinin içeren bir spec agaroz boncukları üzere yoğunlukları değişen çapraz-bağlantılıIFIC çapı 3. Izcilik Otomatik sütun HIC medyanın geleceği, daha ayrıntılı bir optimizasyon denemeleri için istihdam ve protein saflaştırma 4 çalışır gereken belirlemek için etkin bir yaklaşım getirmektedir. Burada saflaştırılmış olan özel protein rekombinant yeşil flöresanlı protein (GFP) &#39;dir, ancak bir yaklaşım, bir veya daha fazla hidrofobik yüzey bölgeleri ile diğer proteinleri saflaştırılması için adapte edilmiş olabilir. GFP UV ışığı 5 maruz kaldığında kendi istikrar, 397 nm&#39;de benzersiz ışık absorbansı tepe, ve floresan nedeniyle yararlı bir model protein olarak hizmet vermektedir. Wild tip GFP içeren bakteri lisatı, yüksek tuz tamponu içinde hazırlanan bir Bio-Rad Çift Geçişli orta basınçlı sıvı kromatografisi sisteme yüklenir ve farklı bir ortam ihtiva HIC HiTrap HIC kolonlara adsorbe edildi. Protein kolonlardan elüt ve in-line ve post-çalıştırma tespit yöntemleri ile analiz edildi. Tampon karıştırma, dinamik örnek döngü enjeksiyon, sıralı colUMN seçimi, çoklu dalga boyu analizi, ve iki fraksiyon eluat toplama deneysel yaklaşımın sistemi ve çoğaltılabilirlik işlevselliği artmıştır.

Watch this Scientific Journal Video about Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification at JoVE.com

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

Proteinin saflaştırılması işleminde kullanılmak üzere uygun hidrofobik etkileşim kromatografısi (HIC) ortam tanımlanması için otomatik bir yöntem sunulmaktadır. Otomatik bir yöntem tampon karışım, dinamik bir örnek enjeksiyon döngüsü, ardışık sütun seçimi, çoklu dalga boyu analizi, ve iki fraksiyon eluat toplama dahil olmak üzere, orta basınçlı sıvı kromatografisi sistemi kullanır.

Protein Saflaştırma Kullanım için Otomatik Hidrofobik Etkileşim Kromatografisi Sütun Seçimi

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

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Research

JoVE Journal

Biology

43.5K Görüntüleme.  Seattle University. Proteinin saflaştırılması işleminde kullanılmak üzere uygun hidrofobik etkileşim kromatografısi (HIC) ortam tanımlanması için otomatik bir yöntem sunulmaktadır. Otomatik bir yöntem tampon karışım, dinamik bir örnek enjeksiyon döngüsü, ardışık sütun seçimi, çoklu dalga boyu analizi, ve iki fraksiyon eluat toplama dahil olmak üzere, orta basınçlı sıvı kromatografisi sistemi kullanır.

Video: Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

Fumarylacetoasetat Hydrolase etki alanı Içeren proteinleri ifade, arıtma, kristalizasyon ve enzim Asdimleri

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

Biyokimya

An Economical and Versatile High-Throughput Protein Purification System Using a Multi-Column Plate Adapter

Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is also a key component in the development of new therapeutics. The evolving era of functional proteomics is fueling the demand for high-throughput protein purification and improved techniques to facilitate this. It was hypothesized that a multi column plate adaptor (MCPA) can interface multiple chromatography columns of different resins with multi-well plates for parallel purification. This method offers an economical and versatile method of protein purification that can be used under gravity or vacuum, rivaling the speed of an automated system. The MCPA can be used to recover milligram yields of protein by an affordable and time efficient method for subsequent characterization and analysis. The MCPA has been used for high-throughput affinity purification of SH3 domains. Ion exchange has also been demonstrated via the MCPA to purify protein post Ni-NTA affinity chromatography, indicating how this system can be adapted to other purification types. Due to its setup with multiple columns, individual customization of parameters can be made in the same purification, unachievable by the current plate-based methods.

A multi-column plate adapter allows chromatography columns to be interfaced with multi-well collection plates for parallel affinity or ion exchange purification providing an economical high throughput protein purification method. It can be used under gravity or vacuum yielding milligram quantities of protein via affordable instrumentation.

Çok Sütunlu Plaka Adaptörü Kullanan Ekonomik ve Çok Yönlü Yüksek Verimli Protein Arıtma Sistemi

Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is ...

Chromatography-based Biomolecule Purification Methods

In biochemistry, chromatography-based purification methods are employed to isolate compounds from a complex mixture. Two such methods used commonly by biochemists are size-exclusion chromatography and affinity chromatography. In size-exclusion chromatography, a column packed with porous beads separates components of a mixture based on size. On the other hand, affinity chromatography allows for a more specific separation of biomolecules by using a column that is composed of stationary phase, which contains target-specific ligands.
This video serves as an introduction to size-exclusion and affinity chromatography, as well as the concepts that govern them. A step-by-step procedure for the purification of a histidine-tagged protein by immobilized metal affinity chromatography is described. Applications for both of these chromatographic methods in biochemistry and biomedical research are also profiled.
<div class="chapter_text" id="script_0">
&#34;Chromatography&#34; refers to a wide range of methods used to isolate a component from a complex mixture, an essential step before a biomolecule&#39;s properties and activities can be determined. Each chromatographic technique has a different mechanism for separation, depending on the sample matrix and target compound. This video will focus on the principles and operation of two methods common to biochemistry: size-exclusion and affinity chromatography.
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<div class="chapter_text" id="script_1">
Size-exclusion chromatography or SEC is based on the size of the compounds in the sample. A mobile phase containing the sample is added to a column with a porous material: the stationary phase. The molecules in the sample fall into 1 of 3 categories.
Molecules too large to enter the pores travel the shortest distance through the column. Any species with a molecular weight above this &#34;exclusion limit&#34; will exit the column at the same time. Molecules small enough to freely enter the pores will be retained the longest, and will exit the column together. The molecular weight allowing complete pore entry is the &#34;permeation limit&#34;.
Only molecules between these limits will be separated from one another, as they spend varying amounts of time diffusing into and out of the pores. Smaller molecules are retained longer on the column because they spend more time in the stationary phase, whereas larger molecules within these limits exit earlier.
Molecular weights across 1 to 2 orders of magnitude fall within these limits. Columns are chosen with this in mind, or multiple columns can be used in series if there is a wide range of desired compounds.
Now that you&#39;ve seen the theory of SEC, let&#39;s look at how it is carried out.
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<div class="chapter_text" id="script_2">
To begin the SEC procedure, the column must be equilibrated with deionized water and chromatography buffer. Once prepared, buffer containing the sample is injected onto the column. The buffer is then pushed through at a low flow rate. A detector monitors what exits the column to determine the presence of the desired analyte. Large molecules with molecular weights above the exclusion limit exit the column at the same time. Small fractions from the column are collected in tubes. Each fraction is tested for the quality of the target molecule by gel electrophoresis or other analytical techniques.
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<div class="chapter_text" id="script_3">
Now let&#39;s have a look at affinity chromatography or AC, one of the most efficient ways to purify proteins. Many biomolecules bind selectively to certain ligands-a property which AC utilizes by adhering a target-specific ligand to the stationary phase.
When the mixture flows through the column, the target molecules attach to the ligand, and the rest flow through. After the mixture has passed through the column, the target molecule can be collected through one of two elution methods based on specificity.
Biospecific elution can be said to a have a &#34;normal-&#34; or &#34;reverse-role&#34;. In normal-role biospecific elution, an agent is added that competes with the adhered ligand to bind with the target biomolecule.
In reverse-role biospecific elution, an agent competes with the target to bind to the adhered ligand. The second elution type, nonspecific elution, lowers the target-to-ligand binding by changing the solution&#39;s pH, ionic strength, or polarity. If a protein does not bind to a ligand that can be immobilized, the protein can be expressed containing a &#34;tag&#34;: short peptide sequences engineered to bind to the ligand.
One variety is immobilized metal ion affinity chromatography, &#34;IMAC&#34; for short, where an adhered metal ligand, like nickel or cobalt, binds to histidine residues on the modified protein. Through molecular biology techniques, target proteins are generated with repeating histidine residues, called a polyhistidine-tag, which binds to the metal via the imidazole side chain on histidine. Once bound, the protein can be collected with free imidazole via reverse-role specific elution and later used in a wide variety of downstream applications.&#160;Now that you&#39;ve seen the theory of affinity chromatography, let&#39;s look at an IMAC procedure in the laboratory.

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<div class="chapter_text" id="script_4">
In IMAC, the stationary phase can be added directly to the mixture of mobile phase and sample, allowing the binding of the his-tagged protein. This slurry is then poured into the column, where the non-bound compounds drip into waste, while the slurry remains. The slurry container is rinsed to collect residual resin and sample, which is added to the column.
The resin is stirred to ensure the unbound components are free-flowing. Added wash buffer helps flush them away. Once all of the unbound components have been removed, the waste is replaced with a container to collect the target protein.
Buffer containing imidazole is added, stirred, and allowed to rest to unbind the target molecule. The imidazole binds to the metal, replacing and releasing the tagged protein. The freed protein is collected, and the imidazole step is repeated to ensure total collection. To further purify the sample, SEC can be run on the sample prior to analysis.
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<div class="chapter_text" id="script_5">
Now that we&#39;ve seen the theory and procedure of these two techniques, let&#39;s look at some of the ways they&#39;re applied in the biochemical field.
A common reason to purify proteins is to study their role in disease. Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator protein, or CFTR. After growing the protein with a his-tag in yeast, both affinity and size-exclusion chromatography allow the isolation of the protein, followed by the study of its function.
In some instances, the presence of a polyhistidine tag can change a protein&#39;s structure, thereby affecting its function. Another common tag is maltose-binding protein or MBP, which will bind to bound amylose in a column. Maltose is then used to release the complex. The MBP can then be cleaved and removed with SEC to produce the pure desired protein.
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<div class="chapter_text" id="script_6">
You&#39;ve just watched JoVE&#39;s video on size-exclusion and affinity chromatography. It covered the theory of the techniques, went over general procedures, and covered some of the uses of the techniques.
Thanks for watching!
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Size Exclusion and Affinity Chromatography

In biochemistry, chromatography-based purification methods are employed to isolate compounds from a complex mixture. Two such methods used commonly by ...

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Kimya

Nickel Affinity Chromatography-Based Protein Purification: A Technique to Purify Polyhistidine-Tagged Recombinant Proteins from Bacterial Cell Lysate

Source: Pokhrel, A. et al. A <a href="https://www.jove.com/v/58547">Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile</a>. J. Vis. Exp. (2018)
In this video, we demonstrate the nickel affinity chromatography technique to purify histidine-tagged pyrophosphokinase enzymes from Clostridium difficile bacteria.

Nikel Afinite Kromatografisi Bazlı Protein Saflaştırması: Polihistidin Etiketli Rekombinant Proteinleri Bakteriyel Hücre Lizatından Saflaştırmak İçin Bir Teknik

Source: Pokhrel, A. et al. A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile. J. Vis. Exp. (2018)
In this ...

JoVE Encyclopedia of Experiments

Biyolojik Teknikler

Kromatografi Teknikleri

Purification of Self-Assembling Protein Nanoparticles using Affinity Chromatography

Source: Karch, C. P. et al., <a href="https://app.jove.com/v/60103">Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation.</a>&#160;J. Vis. Exp. (2019)
This video showcases the purification of histidine-tagged self-assembled protein nanoparticles or SAPNs using affinity liquid chromatography. The resulting purified SAPN fractions are suitable for vaccine development, holding promise for potential immunotherapeutic applications.

Afinite Kromatografisi Kullanılarak Kendi Kendine Birleşen Protein Nanopartiküllerinin Saflaştırılması

1. Expression of the Self-assembling protein nanoparticles core containing a six-helix bundle (SHB-SAPN) Protein in E. coli BL21(DE3)

	Mix 95 mL of ...

İmmünoloji

İmmünoterapi

İmmünojenikler ve Aşı Geliştirme

Butyl-Dependant Hydrophobic Interaction Liquid Chromatography: A Technique to Characterize Antibody-Drug Conjugates Based on Hydrophobic Interactions

Source: Oller-Salvia, B. <a href="https://www.jove.com/v/58066">Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal Reaction</a>. J. Vis. Exp. (2018).
This video demonstrates the hydrophobic interaction liquid chromatography-based characterization of antibody-drug conjugates. Using a butyl ligand-based chromatography column, the antibody-drug conjugates are characterized via hydrophobic interactions with the ligand.

Bütil Bağımlı Hidrofobik Etkileşim Sıvı Kromatografisi: Hidrofobik Etkileşimlere Dayalı Antikor-İlaç Konjugatlarını Karakterize Etmek İçin Bir Teknik

Source: Oller-Salvia, B. Genetic Encoding of a Non-Canonical Amino Acid for the Generation of Antibody-Drug Conjugates Through a Fast Bioorthogonal ...

An Affinity Chromatography Technique for the Purification of Monoclonal Antibodies

Source: Elgundi, Z., et al. <a href="https://app.jove.com/v/55153">Laboratory Scale Production and Purification of a Therapeutic Antibody.</a> J. Vis. Exp. (2017).
This video demonstrates a technique for purifying monoclonal antibodies from the culture supernatant obtained from an antibody-producing cell culture. The process involves using a matrix of crosslinked agarose beads with conjugated Protein A, which immobilizes the antibodies in the supernatant on the beads, allowing for effective separation from contaminants. By applying an elution buffer with low pH, the protein A-antibody interaction is loosened, facilitating the collection of antibody-containing fractions.

Monoklonal Antikorların Saflaştırılması için Bir Afinite Kromatografisi Tekniği

1. Antibody Purification by Affinity Chromatography Using an Automated Fast Protein Liquid Chromatography (FPLC) System
NOTE: The following procedure can ...

Antikor Tabanlı Teknolojiler

Üretim ve Saflaştırma

Types Of Column Chromatography

The stability and compatibility of column material with samples are crucial for efficient purification in chromatographic techniques. Various operating parameters such as pH, temperature, or solvent affect the packing of the column material, thereby determining the purification efficiency. The choice of column material also plays an essential role in deciding the operating parameters and can be modified based on the proteins that need to be purified.
Gel Filtration Chromatography 
When the protein&#39;s chemical nature is unknown, gel filtration chromatography is used for purification based on size. Matrices such as agarose, polyacrylamide, dextran derivatives, or silica beads are used for low-pressure systems, whereas polymeric resins are used for higher flow rates. The varying degree of cross-linking of these polymeric materials determines the pore size of the matrix, which decides the size range of proteins that can be separated.
Ion Exchange Chromatography
Ion-exchange chromatography can purify proteins based on their net charge at a particular pH determined by their isoelectric point or pI. The net charge on matrix resin is used to purify oppositely charged proteins by replacing the cations or anions from the matrix resin with the charged proteins. The resins are categorized based on the type of ion that is exchanged &#8212; positively charged proteins replace cations from cation-exchange resins, and vice versa. Strong ion exchangers with stability over a wide range of buffer pH include quaternary ammonium, sulfonate, and sulfopropyl resins. In contrast, weak ion exchangers that are effective only in a narrow pH range include diethyl aminoethyl (DEAE) or carboxymethyl (CM) resins.
Affinity Chromatography
Affinity chromatography involves immobilizing protein-specific antibodies or enzyme-specific substrates to the column material, usually agarose beads. For example, a column with the immobilized antibody of choice is prepared to separate specific antigens. When the mixed antigen sample is passed through the&#160; column, the target antigens bind to the immobilized antibodies and separate from the mixture. To separate recombinant proteins tagged with enzymes such as beta-galactosidase, the corresponding substrate Isopropyl-&#946;-D-thiogalactopyranoside (IPTG) is immobilized on the column to purify the enzyme.
High-pressure liquid chromatography (HPLC) and gas chromatography (GC) are the most commonly used column chromatography techniques. In HPLC, the solvent flows at high pressure through a tightly packed column that improves efficiency. Gas chromatography analyzes volatile compounds by converting them to their gaseous forms upon heating. The volatilized sample is pushed through the column by inert gasses such as nitrogen and helium.

Kolon Kromatografisi Çeşitleri

The stability and compatibility of column material with samples are crucial for efficient purification in chromatographic techniques. Various operating ...

JoVE Core

Hücre Biyolojisi

Unit 1

Hücrelerin ve Proteinlerin Analizi

KEY TERMS AND CONCEPTS

Hydrophilic Interaction Liquid Chromatography: A Technique to Separate Hydrophilic Polar Analytes Using Hydrophilic Beads

First, open the software to control the mobile phases. Wash UPLC instruments with 50% solvent B and 50% solvent C at the flow rate of 0.2 milliliters per minute, for 30 minutes. Then wash, with 25% solvent A and 75% solvent B at a flow rate of 0.2 milliliters per minute, for 20 minutes, then, at a flow rate of 0.4 milliliters per minute.
Dissolve the labeled N-glycans with 25 microliters of a mixture of 100% acetonitrile and ultra-pure water at a 2:1 (volume/volume) ratio. Then, centrifuge at 134 x g for 5 minutes at 4 degrees Celsius, and use a drive pipe to load 10 microliters of the supernatant into the UPLC instruments.
Separate the labeled N-glycans at a flow rate of 0.4 milliliters per minute, with a linear gradient of 75% to 62% acetonitrile for 25 minutes. Then, perform an analytical run by Dextran Calibration Ladder/Glycopeptide column on a UPLC at 60 degrees Celsius.
Detect N-glycan fluorescence at excitation and emission wavelengths of 330 nanometers and 420 nanometers respectively.

Hidrofilik Etkileşim Sıvı Kromatografisi: Hidrofilik Polar Analitleri Hidrofilik Boncuklar Kullanarak Ayırmak İçin Bir Teknik

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Calcium-Dependent Hydrophobic Interaction Chromatography: A Technique to Purify Calcium-Binding Proteins Based on Hydrophobic Interactions

To continue the protein purification, after the protein dialysis, add calcium chloride to the sample to a final concentration of 25 millimolar, which will facilitate binding of S100A12 to the resin in the next step. Then, filter the sample through a filter with a pour size of 0.45 microns.
Equilibrate HIC buffers and samples to 4 degrees Celsius. Next, connect column buffers HIC-A and B and the column, to the system and adjust the parameters.
Start the method to equilibrate the column with 1 to 2 column volumes of buffer HIC-A, and load the sample, and the &#39;wash unbound sample block&#39; when the UV signal reaches baseline level again.
Then, start elution with a calcium chelator-containing buffer EDTA. Collect 2 milliliters of peak fractions during elution.

Protein Saflaştırma Kullanım için Otomatik Hidrofobik Etkileşim Kromatografisi Sütun Seçimi

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