Bu proje, Knut ve Alice Wallenberg Vakfı ve İsveç Araştırma Konseyi tarafından finanse edildi.

1. Klonlama inşa hedef gene/ABDz1-tagged füzyon <ol><li> Ifade plazmid hedef gen (plazmid içeren ABDz1 bir malzeme transferi anlaşması üzerinden serbestçe kullanılabilir) N-terminal ligasyonu için uygun kısıtlama siteleri tarafından çevrili ABDz1 geninin PCR parçaları hazırlayın. </li><li> Cleave saflaştırılmış ifade vektör ve uygun bir reaksiyon tamponu seçilen restriksiyon enzimleri ile PCR parçaları. Ligasyonu önce ürünleri arındırın. </li><li> , Ilgi gen içeren ekspresyon vektörü sınırlı ABDz1 parçası bağlanır ve E. ligasyonu ürün dönüşümü coli (orijinal yöntem RR1ΔM15 zorlanma 8 kullanılmıştır). Seçim için uygun antibiyotikler ile takviye agar plaklarına dönüştürülmüş hücreleri yayılmıştır. </li><li> PCR ekran birkaç koloni ve sıra DNA dizi analizi ile ortaya çıkan ifade kaset doğrulayın. </li><li> Bir gecede bir dizi verif kültürü plazmid hazırlayınIED koloni ve tercih edilen bir ifade suşu (Rosetta insan genleri tarafından kodlanan proteinlerin üretimini artırmak için plazmid bir pRARE barındıran özgün yöntemi E. coli (de3) kullanılmıştır) dönüşüm . </li></ol> 2. Protein ekspresyonu <ol><li> Tek bir bakteri kolonisi, 5 g / l maya özü ve uygun antibiyotik ile desteklenmiş triptik soy broth 10 ml inoküle edin. ° C gecede 37 150 rpm&#39;de inkübe edin. </li><li> 1 ml, 100 ml taze orta gecede kültür aşılamak ve hücrelerin logaritmik büyüme fazı ulaştığınızda protein ekspresyonu (lac operonunun kullanılan izopropil-β-D-thiogalactoside başlangıçta 1 mM) neden. </li><li> 150 rpm&#39;de 25 ° C gecelik ve hasat hücreler santrifüj tarafından inkübe edin. </li></ol> 3. Ortogonal yakınlık arıtma <ol><li> 25 ml çalıştıran tamponu (25 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA,% 0.05 (w / v) Tween 20, pH 8.0) pelletini tekrar ve th bozabilir% 60 genlik ve 3 dakika 1.0/1.0 bakliyat sonication e hücreleri. Örnek Santrifüj ve supernatant (0,45 mikron) ve daha ileri saflaştırma önce hedef protein içeren filtre. </li><li> 1 ml / dak &#39;da uygun bir protein arıtma sistemi tampon çalışan 10 sütun hacimleri (CV) ile tedarikçi tavsiyelerine göre daha önce HSA ile birleştiğinde ya da 1 ml HSA sepharose kolon veya NHS aktif sütun, muvazene. 0.5 ml / dak bakteriyel lizat yükleyin ve sonra akış hızı 1 ml / dak sıfırlanır. </li><li> 5 yıkama tampon CV (5 mM NH 4 Ac, pH 5.5) takip çalışan tampon CV ile 5 sütun yıkayın. </li><li> 1 ml / dak elüsyon tamponunda (0.5 M HAC, pH 2.5) ile örnek Zehir ve daha ileri saflaştırma için yıkandı, zirveden kesirler seçmek için 280 nm, monitör emme kesirleri toplamak. </li><li> Yüksek protein konsantrasyonu Havuz kesirler, Sure çalışan tampon iki kez seyreltilmiş (20 mM fosfat, 150 mM NaCl, pH 7.2) vepH yaklaşık nötr olduğundan emin olun. Gerekirse pH artırmak için 1 M Tris-HCl, pH 8 ekleyin. </li><li> 0.5 ml / dk, 1 ml / dak Sure çalışan bir tampon 10 CV ile dengelenmiş bir 1 ml HiTrap MabSelect Sure sütun örnek yükleyin. Yükledikten sonra 1 ml / dak akış hızı sıfırlayın. </li><li> Tabii çalışan tampon 5 CV (adım 5) sütun yıkayın ve 0.2 M HAC, pH 2.7 ile proteinlerin Zehir. Hassas hedef proteinlerin eluat toplama üzerine Tris-HCl ilavesi ile doğrudan nötralize edilebilir. </li></ol> Kromatografik adımlar MabSelect eluat tersine çevrilirse Sure sütun tampon (adım 3.1) çalışan seyreltilir ve 1 M Tris-HCl ilave edilerek pH yaklaşık 8 artış olabilir. Emin matris ikinci adımı istihdam Genel olarak, dar doruklarına görülmektedir. 4. Sodyum dodesil sülfat poliakrilamid jel elektroforezi (SDS-PAGE) ile saflık Değerlendirme <ol><li> Yük bir saflaştırılmış fraksiyonlarıeğer istenirse, kütle spektrometresi ile analiz arındırılmış ürün molekül ağırlığı, SDS-PAGE azaltılması ve. ABDz1 ücretsiz bir sistein düşürücü olmayan koşullar altında ürün dimerization neden olabilir bu yana tam örnek azaltmak için önemlidir. </li></ol> 5. Temsilcisi Sonuçlar: Ilkesinin bir kanıtı olarak, dik yakınlık arıtma ABDz1 etiketi ile kolaylaştırdı farklı çözünürlük sınıfları, moleküler ağırlık ve izoelektrik noktaları (Tablo 1) temsil eden üç insan hedef proteinlerin değerlendirildi. ABDz1 gen, genetik, hedef genlerin erimiş ve yapıları E. coli ifade edildi, Şekil 2 ardışık yöntemi tüm adımlar için bir akış şeması gösterir. Dik protokolü tarafından arıtma ardından, farklı noktalarda toplanan numuneler, SDS-PAGE (Şekil 3) ile analiz edildi. Sonuçları bir çift etiket ve iki son derece özel bir arıtma adımları yararını açıkça göstermektedir. Makul bir saflık ac olsa bilehieved jeller görüldüğü gibi ilk arıtma adımdan sonra, ardışık adım de son derece zorlu uygulamalar için uygun, çok saf bir ürün elde edilir. <img alt="Şekil 1" src="/files/ftp_upload/3370/3370fig1.jpg" /> Şekil 1 bispecific ABDz1 etiketi seçimi için kullanılan Kombinatoryal kütüphane tasarımı. 46 amino asit albümin bağlayıcı etki alanı istikrarlı bir üç sarmal paket içine kıvrım ve özellikle ikinci sarmal bulunan insan serum albümin için bağlayıcı bir site içerir. Genetik on birinci ve üçüncü sarmal bulunan yüzey maruz kalan amino asitler rastgele olarak, yeni bir bağlayıcı yüzey mühendisliği oldu. Onbir pozisyonları rakam gösterilir ve Kraulis ve ark göre numaralandırılmıştır. [9 ]. Faj üzerinde ifade Kombinatoryal kütüphanesi ile, bispecific ABDz1 molekül tespit edildi Stafilokokal protein Z etki alanı bir dimer karşı biopanning ardından. <img alt = &quot;Şekil 2&quot; src = &quot;/ files/ftp_upload/3370/3370fig2.jpg&quot; /&gt; Şekil 2. dik benzeşme arıtma protokolü için basitleştirilmiş akış şeması. ABDz1 dizisi içeren bir PCR-parçası, uygun bir ifade vektör ilgi bir gen ile (N-terminal) bağlandı. Ligasyonu ürün E. dönüştürülmüştür. sıra doğrulama ve plazmid hazırlık için coli. Bir ifadesi ev sahibi dönüşümün ardından, rekombinant protein ekspresyonu için büyük ölçekli bir kültür, bir ilk gecede kültür kurulur. Bakteriler santrifüj yoluyla, hedef protein içeren bakteriyel lizat üretmek için hasat ve sonication tarafından parçalanmış. Artık katı parçacıklar kaldırmak için bir filtrasyon adımdan sonra art arda iki arıtma adımları geçiren lizat bir sıvı taşıma sistemi dik yakınlık arıtma tabi tutulur. Yıkandı, hem de arıtma adımları doruklarına örneklenmiş ve saflık değerlendirmek için SDS-PAGE tarafından değerlendirilir ve t karşılaştırıldığındao arıtma önce lizat. <img alt="Şekil 3" src="/files/ftp_upload/3370/3370fig3.jpg" /> Şekil 3. füzyon ifade ABDz1 ile saflaştırılmış hedef proteinleri SDS-PAGE analizi. Sure-sütun MabSelect tarafından izlenen bir HSA-sütun üzerinde arıtma karşılık gelen doruklarına örnekler, farklı özellikler gösteren ABDz1, ABDz1 Etiketin kendisi için füzyon ifade üç proteinlerin bakteriyel lizat alınan numuneler ile bir araya toplanmıştır (A) . Lanes 1-4 9-12 MabSelect Sure arıtma (ikinci adım) arıtma, HSA arıtma şerit 5-8 (ilk adım) ve şerit önce lizatları örnekler temsil eder. ABDz1-141.377, ABDz1-HT875, ABDz1-HT2375 ve ABDz1: Örnekler aşağıdaki sırayla yüklenir. (B) yanı sıra, aynı sütun üzerinde tersten saflaştırılmış aynı lizatları elde örnekleri incelendi. Lanes arıtma, şerit 4-6 f önce lizatları örnekleri temsil 1-3rom MabSelect emin arıtma (ilk adım) ve HSA arıtma (ikinci adım) şerit 7-9. Örnekleri (A), ancak etiket kendisi dahil değildir olarak aynı sırayla yüklendi. Bu sonuçlardan yola çıkarak bu iki adımlı bir yöntem adımları sırayla uygulanır bakılmaksızın yüksek derecede saflaştırılmış protein verir açıktır. <table border="1"><tbody><tr><td> Adı b </td><td> Hedef protein Uniprot c </td><td> Moleküler ağırlık (kDa) </td><td> Çözünürlük sınıf d </td><td> Molekül ağırlığı füzyon ürünü (kDa) </td><td> Izoelektrik nokta füzyon ürün </td></tr><tr><td> 141377 </td><td> B7Z315 </td><td> 17,4 </td><td> 4 </td><td> 23,7 </td><td> 8,5 </td></tr><tr><td> HT875 </td><td> P01040 </td><td> 10,8 </td><td> 3 </td><td> 17,1 </td><td> 4,9 </td></tr><tr><td> HT2375 </td><td> P00740 </td><td> 8,9 </td><td> 5 </td><td> 15,2 </td><td> 6,8 </td></tr></tbody></table><ol class="lalpha"><li> Yalnız ABDz1 etiketi Molekül ağırlığı 6.3 kDa, izoelektrik noktası 6.7. </li><li> Hedef protein, Uniprot protein bir kısmını temsil eder. </li><li> <a href="http://www.uniprot.org">http://www.uniprot.org</a> . </li><li> N-terminal protein parçası Onun 6 ABP 10 Çözünürlüğü sınıfı. </li></ol> Tablo 1. ABDz1 etiketi ile hedef proteinler ve füzyon ürünleri bir ilke çalışmanın bir kanıtı olarak değerlendirilir. Üç farklı molekül ağırlığı, çözünürlük ve izoelektrik noktası eşsiz insan proteinleri dik benzeşme yaklaşımı ile ifade ve saflaştırılması için seçilmiştir.

<ol>
	<li>Waugh, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+the+most+of+affinity+tags.">Making the most of affinity tags.</a> Trends Biotechnol. 23, 316-320 (2005).</li><li>Hedhammar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+engineering+strategies+for+selective+protein+purification.">Protein engineering strategies for selective protein purification.</a> CET. 28, 1315-1325 (2005).</li><li>Porath, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal+chelate+affinity+chromatography,+a+new+approach+to+protein+fractionation.">Metal chelate affinity chromatography, a new approach to protein fractionation.</a> Nature. 258, 206-213 (1975).</li><li>Linhult, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutational+analysis+of+the+interaction+between+albumin-binding+domain+from+streptococcal+protein+G+and+human+serum+albumin.">Mutational analysis of the interaction between albumin-binding domain from streptococcal protein G and human serum albumin.</a> Protein Sci. 11, 206-213 (2002).</li><li>Nilsson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+synthetic+IgG-binding+domain+based+on+staphylococcal+protein.">A synthetic IgG-binding domain based on staphylococcal protein.</a> A. Protein Eng. 1, 107-113 (1987).</li><li>Alm, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small+bispecific+protein+selected+for+orthogonal+affinity+purification.">A small bispecific protein selected for orthogonal affinity purification.</a> Biotechnol. J. 5, 605-617 (2010).</li><li>Nilsson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+affinity+domains+for+the+detection,+purification+and+immobilization+of+recombinant+proteins.">Multiple affinity domains for the detection, purification and immobilization of recombinant proteins.</a> J. Mol. Recognit. 9, 585-594 (1996).</li><li>R&#252;ther, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pUR+250+allows+rapid+chemical+sequencing+of+both+DNA+strands+of+its+inserts.">pUR 250 allows rapid chemical sequencing of both DNA strands of its inserts.</a> Nucleic Acids Res. 10, 5765-5772 (1982).</li><li>Kraulis, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+serum+albumin-binding+domain+of+streptococcal+protein+G+is+a+three-helical+bundle:+a+heteronuclear+NMR+study.">The serum albumin-binding domain of streptococcal protein G is a three-helical bundle: a heteronuclear NMR study.</a> FEBS Lett. 378, 190-194 (1996).</li><li>Hedhammar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+flow+cytometry-based+method+for+analysis+of+expression+levels+in+E+coli+giving+information+about+precipitated+and+soluble+protein.">Novel flow cytometry-based method for analysis of expression levels in E coli giving information about precipitated and soluble protein.</a> J. Biotechnol. 119, 133-146 (2005).</li><li>Carter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+proteolysis+of+fusion+proteins.">Site-specific proteolysis of fusion proteins.</a> ACS Sympos. Ser. 427, 181-193 (1990).</li><li>Hedhammar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negatively+charged+purification+tags+for+selective+anion-exchange+recovery.">Negatively charged purification tags for selective anion-exchange recovery.</a> PEDS. 17, 779-786 (2004).</li><li>Gr&#228;slund, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charge+engineering+of+a+protein+domain+to+allow+efficient+ion-exchange+recovery.">Charge engineering of a protein domain to allow efficient ion-exchange recovery.</a> Protein Eng. 13, 703-709 (2000).</li></ol>

Çıkar çatışması ilan etti.

Burada sunulan dik benzeşme arıtma protokolü hedef proteinlerin geniş bir yelpazede etkili arınma sağlar. Bağlayıcı bir roman yüzeyi ile albümin bağlayıcı etki doğasında bağlayıcı site birleştirerek, küçük bir bispecific protein etiketi geliştirilmiştir. Bu, sadece herhangi bir tercih ifade vektör ilgi herhangi bir protein klonlanmış ve füzyon ifade beri etiket kullanmak için çok basittir. Çoğu laboratuvarlarda kullanılabilir Standart donanım, iki adımlı bir protein saflaştırılması için istihdam edilebilir. ABDz1 etiketi etki alanı verimli bir şekilde iki bağlayıcı yüzeyler ortaya çıkarmak için doğru katlama fonksiyonu bağlı olduğundan, bu yöntem çözünür şeklinde ifade proteinler sınırlı ve denatüre koşulları altında saflaştırılması için uygun değildir. Indirgeyici ajanlar, MabSelect emin bir matris üzerine Z-etki alanına ABDz1 bağlama ile müdahale yılından bu yana da kaçınılmalıdır. Ortogonal afinite purificaTION birçok zorlu uygulamalar proteinlerin yüksek oranda saflaştırılmış gereksinimlerini karşılamak için geleneksel yöntemlere bir alternatif sağlar. Bu yöntem aynı zamanda, farklı saflaştırma stratejileri arkaya kullanılan Kombinatoryal etiketleme ihtiyacını ortadan kaldırır. Yerli bir hedef protein gerektiren uygulamalar için, mümkün ABDz1 etiketi 11 enzimatik kaldırılmasını işlemek için proteaz bölünme sitesi tanıtıldı olabilir. Ayrıca, bugüne kadar açıklanan ilk bispecific küçük ve katlanmış protein etki alanı ABDz1 etiketi. Iki hedef özel yakınlık etkileşimleri bağlıdır arıtma stratejisi sağlar çünkü benzersizdir. Geleceğe hazır ve ucuz kromatografik reçine ile uyumlu olan yeni bağlayıcı yüzeyler taşıyan roman bispecific etiketleri gelişmekte olan bu konsepti genişletmek için ilginç olacaktır. Örneğin, temel kalıntıları, bir etiketi i ile uyumlu amino asitler albümin bağlayıcı bir yer değiştirerekdeğiştirici kromatografi ulaşılabilir olabilir. Benzer bir kavram, sırasıyla, Z asit 12 ve Z temel 13 anyon ve katyon değişim ile uyumlu olarak bilinen etiketleri oluşturmak için Z-etki sorumlu mühendislik etkili olduğu kanıtlanmıştır .

<table border="1"><tbody><tr><td> Reaktifi Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> E. coli RR1ΔM15 </td><td> Amerikan Tip Kültür Koleksiyonu </td><td> 35102 </td></tr><tr><td> Rosetta E. coli (de3) </td><td> Novagen </td><td> 70954 </td></tr><tr><td> Triptik soy broth </td><td> Difco </td><td> 211822 </td></tr><tr><td> Maya özü </td><td> Difco </td><td> 212720 </td></tr><tr><td> Titreşim hücre sonikatör </td><td> Sonics ve malzeme </td><td> - </td></tr><tr><td> HSA sepharose </td><td> Pharmacia Biotech </td><td> Eski ürün </td></tr><tr><td> Tabii HiTrap MabSelect </td><td> GE Healthcare </td><td> 11-0034-93 </td></tr><tr><td> HiTrap NHS aktive HP sütun </td><td> GE Healthcare </td><td> 17-0716-01 </td></tr></tbody></table>

orthogonal protein purification facilitated by a small bispecific affinity tag

Protokolleri rasyonalize gerekir rekombinant proteinlerin saflaştırılması ile ilgili yüksek maliyetler nedeniyle. Yüksek verimli çalışmalar için hedef protein özel optimizasyon 1 gerekmez genel yöntemler için bir talep vardır. Bunu başarmak için, genetik ilgi gen erimiş olabilir arıtma etiketleri genellikle 2 kullanılır. En yaygın olarak kullanılan bir yakınlık kolu hexa-histidin etiketi, yerli ve denatüre edici koşullar 3 hem de altında arıtma için uygundur . Etiketiyle üretimi için metabolik yükünün düşük, ama rakip afinite kromatografisi dayalı stratejiler 1,2 gibi yüksek bir özgüllük olarak sağlamaz . Burada, 46 amino asit iki farklı bağlanma yerleri ile bispecific arıtma etiketi, küçük protein etki alanı olmuştur. Albümin bağlayıcı etki alanı Streptokoksik protein G türetilmiştir ve insan serum albümin güçlü bir doğal yakınlığına sahiptir.Kalkış Desteği (HSA). Onbir yüzey maruz albümin bağlayıcı 4 ilgisi olmayan amino asitler, kombinatoryal bir kütüphane üretmek için genetik olarak randomize edildi . Rastgele bağlayıcı yüzey (Şekil 1) düzenlenen romanı ile protein kütüphane faj ekran teknolojisi bağlayıcı seçimi kolaylaştırmak için faj partikülleri ifade edildi. Dimerik karşı biopanning birkaç tur sayesinde Z Stafilokokal protein A 5, HSA ve roman hedef 6 tespit edildi hem yakınlığa sahip küçük bir bispecific molekül türetilmiş etki alanı. ABDz1 olarak anılacaktır roman protein etki, farklı molekül ağırlığı, çözünürlük ve izoelektrik nokta ile hedef proteinlerin bir seçim için bir arınma etiketi olarak değerlendirildi. Üç hedef proteinlere Escherishia coli K-Termini erimiş ve daha sonra afinite saflaştırılmış roman etiketiyle ifade edildi. Immobilize HSA veya Z-etki alanı ya da bir sütun ilk arıtma ilişkiler sonuçlandıvely saf ürünler. Bispecific etiketi ile iki adım yakınlık arıtma protein saflık önemli bir iyileşme sonuçlandı. Kromatografik Z-alanıyla medya, Sure örnek MabSelect, immobilize antikor saflaştırılması için hazır ve HSA kolayca kimyasal ikinci matris sağlamak için medya birleştiğinde olabilir. Kurtarılan hedef proteinin saflığı yüksek bir talep olduğunda bu yöntem özellikle avantajlıdır. Ifade ev sahibi metabolik yük etiketiyle küçük boyutu nedeniyle sınırlı iken etiketi bifunctionality iki farklı kromatografik adımları kullanılmasına izin verir. Birden fazla etiket kombinasyonu 1,7 kullanılır Kombinatoryal etiketleme denilen rekabetçi bir alternatif sunuyor.

Watch this Scientific Journal Video about Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag at JoVE.com

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Yeni ve son derece verimli, iki kademeli afinite kromatografisi protokolü geliştirildi ve ayrıntılı olarak açıklanmıştır. Bu yöntem iki içsel yakınlık küçük bir arıtma etiketi dayanan ve farklı özelliklere sahip geniş bir hedef proteinlerin uygulanabilir.

Küçük Bispecific Affinity Tag Kolaylaştırılmış Ortogonal Protein Saflaştırma

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

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16.9K Görüntüleme.  Royal Institute of Technology. Yeni ve son derece verimli, iki kademeli afinite kromatografisi protokolü geliştirildi ve ayrıntılı olarak açıklanmıştır. Bu yöntem iki içsel yakınlık küçük bir arıtma etiketi dayanan ve farklı özelliklere sahip geniş bir hedef proteinlerin uygulanabilir.

Video: Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Organik Solvent ve Asetik Asit olmadan Jeller Coomassie G-250 ile Proteinlerin Boyama

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Biyoloji

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

Dayanıksız Multi-protein Kompleksleri izolasyonu In vivo Kontrollü Hücresel Çapraz Bağlama ve immuno-manyetik Afinite Kromatografisi

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Hsp104 saflaştırılması, Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

Tandem Affinity Arıtma kullanarak Protein etkileşim Ortaklar belirlenmesi

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Protein Kompleksi belirlenmesi<em&gt; Escherichia coli</em&gt; Tandem Kütle Spektrometresi ile Kombine Sıralı Peptid Affinity Arıtma kullanarak

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

Affinity etiketli Rekombinant Proteinler Yüksek throughput Arıtma

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness1. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST)2,3 and a C-terminal 10xHis tag4, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression5. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme6. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest.

In the present protocol, we demonstrate a highly efficient and cost-effective small-scale protein purification method, which allows purification of recombinant proteins by uniquely combining a cleavable GST-tag and a small His-tag.

GST-His saflaştırma: Tam boy Saflaştırılmış Proteinler Verim iki aşamalı bir saflaştırma protokolü Affinity

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

Mikro Termoforosis tarafından Bağlama Affinity belirlenmesi Protein Saflaştırma-ücretsiz Yöntemi

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

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 ...

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

Protein-protein etkileşimi belirlenmesi<em&gt; Drosophila</emTandem Affinity Arıtma tarafından&gt; Adult Başlar (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...