Biz, onların tavsiyelerini ve yorumlar için verdiği destek ve Geula Davidov, Noam Grimberg ve Chen Guttman Dr. Amir Aharoni kabul etmiş sayılırsınız.

1. E. Mama Gen Klonlama ve İfade coli 5&#39;-GCATTACGCATATGGACGACATCCGCCAGGTG-3 &#39;ve 5&#39;-GCGCGGCAGCCATA-TGGCATACG-3&#39; mutant gen mamAΔ41 primerler Magnetospirillum magneticum AMB-1 genomik DNA polimeraz zincir reaksiyonu (PCR) ile amplifiye edildi. Çoğaltılan DNA parçaları, bir NcoI sitesinde başlatma kodon ATG sunuldu ve fesih kodon bir ScoI site ile değiştirilir. Parçaları NcoI ve Sacl ile sindirilir ve pET52b ilgili siteler (+) klonlanmış pET52bMamAΔ41-AMB1 sebebiyet veren. Bu yapı, mamAΔ41 gen-C-terminalindeki 10-tag ile çerçeve içinde erimiş oldu. Plazmid, E. coli suşu BL21 içine electroporated oldu. E. coli pET52bMamAΔ41 barındıran BL21 (15), oto-indüksiyon 3 saat ampisilin (50 mg / ml), 310 ° K içeren orta yetiştirilen süzün . Ekimi sıcaklığı 310 ° &#39;den 300 ° K, 300 ° K. kaydırılır ve ek bir 48 saat süreyle devam edildi Hücreleri 277 az 10 dak ° K. 5465g santrifüj tarafından toplandı 8 litre kültür ıslak hücre pelet 60 gram üretti. 2. Biyoinformatik Hesaplamalar ProtParam sunucusu (http://www.expasy.ch/tools/protparam.html) kullanarak molekül ağırlığı (MW), amino asitler sırasına göre ve 280nm protein, 1mg/1ml öngörülen emme Hesaplamalar . 10His-Tag-MamAΔ41 için 22529 MW Da (240 amino asitler) ve MamAΔ41 (Trombin tarafından O&#39;nun etiketi kaldırma sonra kalan 9 amino asitler) Mw 20596.5Da (187 amino asitler). 10His-Tag-MamAΔ41 için protein 280nm 1mg/1ml tahmin emme 0,595 ve 0,579 MamAΔ41 için. Buna ek olarak, amino asit dizisi, bir sistein kalıntıları içermez. Bu nedenle, indirgeyici ajanlar arıtma işlemi sırasında gerekli değildir. 3. MamAΔ41, saflaştırılması <ol><li> Stok solüsyonu hazırlanması için, tartmak ve aşağıdaki konsantrasyonlarda (200 ml) hazırlayın: imidazol (MW 228,2 g / mol) 4M çözümü, NaCl (Mw 58,44 g / mol) 5M çözüm, Tris-HCl (Mw 121.3 g / mol ) 1M çözelti, pH = 8 ayarlayın. Çözüm netleşene kadar bir girdap kullanarak çift distile su (DDW) ve karışımı ekleyin. 0.22μm oda sıcaklığında tutmak filtre ve filtre çözümleri. </li><li> Hazırlayın ve stok solüsyonları (Tablo 1) 6 taze tamponlar filtre. </li><li> Seyreltilir ve tampon 1:2 oranını, hücre gram tampon hacmi (ml) hücreler askıya. </li><li> DNaz I (1 mg / ml) bakteriler her 10gr için örnek DNA parçaları kırmak için 10μl ekleyin. EDTA ücretsiz proteaz inhibitörü kokteyl bakterilerin her 20gr için 1 ml ekleyin. 20 dakika süreyle buz üzerinde inkübe edin. </li><li> Fransız basını, iki kür 25.000 psi hücreleri bozulur. Sonication aksine, Fransız basını, ince bir borudan yüksek basınç altında hücreleri zorlayarak yüksek hacimli (&gt; 10 ml) hücre ekstrüzyon hedeflenmektedir. Piston sıcaklık birikimi nedeniyle kullanım ve montaj önce on dakika boyunca buz üzerinde soğutulmalıdır. </li><li> 25ml ultrasantrifüjdeki tüpler içine parçalanmış hücreleri transferi ve örnek tüp boyun hattı ulaşıncaya kadar tampon A (50mg) tam denge. </li><li> 277, 1 saat süreyle 270.000 g Ultra santrifüj ayrı hücre artıkları ° K </li><li> 4 ml% 20 etanol konserveler Ni NTA reçine, sütun üzerine ekleyerek ev yapımı bir ağırlık Ni NTA sütun (2.5cm çap) hazırlayın. Tamamen sıvı bırakalım, daha sonra 40 ml DDW ile yıkayın ve tamamen sıvı bırakalım. 40 ml tampon A. sütun öncesi dengelenmiş reçine </li><li> Yerçekimi Ni NTA sütun üzerinde ultra santrifüj tüpleri çözünebilir fraksiyon uygulayın. Akış yoluyla toplamak (bağlantısı çözülmüş proteinler) ve 277K az korumak, sıvı bırakalım. </li><li> Pelet hurda küçük bir ipucu kullanarak ultra santrifüj tüpleri pelet örnek toplayın. 50μl 5% 2X β-merkaptoetanol SDS-PAGE örnek tampon ucu karıştırın. </li><li> 100 ml tampon B. sütun akış yoluyla toplamak ve 277K az korumak, sıvı bırakalım yıkayın. </li><li> 15 işaretli Eppendorf tüpleri (1.5ml) hazırlayın. </li><li> Elüsyon - yakın sütun vana ve sonra sütun üzerine tampon C 1ml yük, 3 dakika supap açılma ve flow-through toplama takip boyunca inkübe. Bu adımı üç kez tekrarlayın. </li><li> Zehir geri kalanını askıya alma 1 ml tampon C artışlarla ve vanasını kapatın. </li><li> Kesirler 280nm az OD ölçün ve protein zirve konumunu değerlendirmek, OD hala&gt; 0.3 ise, ek örnekler ihtiyaç vardır. </li><li> Toplanan fraksiyonları SDS-PAGE analizi. Böyle bir analiz, aşağıdaki örnekleri ile de yapılmalıdır bağlanmamış proteinler, flow-through-through akış yıkayın ve uLTRA santrifüj pelet örnek. 10μl 5% 2X SDS-PAGE örnek tampon β-mercaptoethanol her örnekten 10μl karıştırın. % 15 SDS-PAGE numuneleri yükleyin ve 180 Volt 45 dakika çalıştırın. </li><li> InstantBlue çözüm 15 ml jel Lekesi, 1 saat süreyle çalkalayın. Plastik kutu kapalı ve ışıktan korumalı olduğunu emin olun. </li><li> Jel değerlendirin; protein ekspresyonu gösterge bantlara göre, yüksek ve özel ise, seçilen yıkandı, kesirler birleştirmek. </li><li> 10-His-etiketi kaldırmak için, kombine örnek için üreticinin tavsiyesine göre, sığır trombin (10 adet / ml), 1 mg protein başına 10μl trombin. </li><li> Diyaliz aşırı imidazol çıkarılması için kullanılır ve iyon değişimi bağlanma sırasında gerekli seviyeleri için NaCl konsantrasyonları azaltmak için. Diyaliz için 7 kD molekül ağırlığı, istenilen uzunlukta kesilmiş (MWCO) diyaliz tüp kesti. DDW ile yıkayın ve bir ucunu (veya klipler ile mühür) bir düğüm. Üst kısmında biraz boşluk bırakarak çanta üzerine birleştirilmiş protein çözüm transferi ve mengene. </li><li> Batmak 4 litre soğuk tampon diyaliz diyaliz tüp (Tampon D). 277 gece boyunca yavaş yavaş çözüm karıştırın ° K </li><li> Beher diyaliz tüp dışarı atın, küçük bir kesim yapmak ve tüp 50ml protein transfer. </li><li> İyon-değişim kromatografisi: Hızlı Performans Sıvı Kromatografisi (FPLC) MonoQ 4.6/100 PE sütun önceden paketlenmiş monte edin. DDW filtrelenmiş su 5 sütun hacimleri (CV) ile sütun yıkayın. Tampon D. 5 CV ile dengelenmiş sütun </li><li> DDW (iki döngü hacimleri) ile enjeksiyon döngü yıkayın ve Yük enjeksiyon döngü içinde protein tampon D. ile tekrarlayın. </li><li> Protein örneği (1 ml / dak akış hızı) enjekte edilir ve sınırsız proteinlerinin SDS-PAGE tespiti için kesirleri toplamaya başlar. </li><li> Tüm örnek sütun yükleninceye kadar, ilk protein hacmi (adım 3.23) enjeksiyon döngü daha büyükse, döngü yıkamadan, 3,25-3,26 adımları tekrarlayın. </li><li> Sınırsız proteinlerin ortadan kaldırmak için 3 CV ile tampon C sütunu yıkayın. </li><li> Tamponlar D &amp; E (% 100 tampon E degrade sonuna), akış hızı 1 ml / dk (arasında 40 2000mm NaCl 60 dakika doğrusal bir degrade, maliyeti yüksek debilerde kullanılan olmasına rağmen protein Zehir geniş hacimli, ama daha az yoğun, protein zirveleri). 280nm kromatogram göründükleri gibi proteinler doruklarına toplayın. </li><li> Temizlik öncesi dolu sütun tavsiye üretimi göre. </li><li> Toplanan fraksiyonları SDS-PAGE analizi. Böyle bir analizi aşağıdaki örnekleri ile yapılmalıdır, sınırsız proteinler akış yoluyla ve aracılığıyla protein doruklarına akış. SDS-PAGE çalıştırın ve adımları 3,17-3,18 söz leke. </li><li> Protein ön lekeli işaretine göre, tahmin edilen Mw protein bantları varlığı için jel değerlendirin. Birleştirme ilgili protein içeren seçilen elüsyon kesirler. </li><li> Boyut dışlama kromatografisi için gerekli NaCl konsantrasyonlarının azaltılması amacıyla, birleştirilmiş numunelerin Dialyze. 3,21-3,23 tampon F. karşı adım belirtildiği gibi diyaliz gerçekleştirin. </li><li> Protein örnek Konsantre bir Vivaspin-15 10.000 MWCO için 8 mg / ml &#39;bir tablo santrifüj, 4000 rpm. Kuvars küvet 280nm az OD ölçerek arzu konsantrasyon onaylayın. Protein çok konsantre ise tampon F sulandırmak ve tekrar ölçmek. </li><li> Boyut dışlama önceden paketlenmiş sütun, FPLC Hiload 26/60 superdex 200, birleştirin; sütun DDW filtrelenmiş su 3 CV ile yıkayın. Tampon F. 3 CV ile dengelenmiş sütun </li><li> Yük en fazla 4 ml enjeksiyon döngü içinde protein örnek enjeksiyon döngü ile aynı hacimde tampon F. takip 5ml DDW (iki döngü cilt) ile yıkayın. </li><li> Protein örneği (3.5 ml / dak akış hızı) enjekte edilir ve 280nm kromatogram göründükleri gibi proteinler zirve kesirleri toplamaya başlar. Sütun hacmi ulaşıncaya kadar tampon akışı olsun. </li><li> Tüm örnek sütun yükleninceye kadar, ilk protein hacmi (adım 3.23) enjeksiyon döngü daha büyükse, döngü yıkamadan, 3,25-3,26 adımları tekrarlayın. </li><li> Temizlik önceden paketlenmiş sütun üretimi önerisine göre. </li><li> Toplanan fraksiyonları SDS-PAGE analizi. Böyle bir analiz protein doruklarına örnekleri ile yapılmalıdır. SDS-PAGE çalıştırın ve adımları 3,17-3,18 söz leke. </li><li> Jel Değerlendirin: protein doruklarına özgü olmadığını, doğru tahmin Mw protein lekeli öncesi marker ve sadece tek bir bant göre ilgili protein yıkandı, kesirler seçili birleştirme görünür. </li><li> &gt; 20 mg / ml, bir tablo santrifüj 4000 rpm bir Vivaspin-15 10.000 MWCO kullanarak protein örnek Konsantre. 280nm az OD ölçerek arzu konsantrasyon onaylayın. Saflaştırılmış MamAΔ41 sonra kristalleşmesi için 26.5 mg / ml konsantre oldu. </li> &lt;li&gt; Matriks yardımlı lazer desorpsiyon / iyonizasyon (MALDI-TOF) kullanarak örnek saflık ve protein kimlik onaylayın. </li><li> Her 25-50μl protein, protein, kütle spektrometresi ve SDS-PAGE göre arıtılmış ise, önceden işaretlenmiş Eppendorf tüplerine konsantre MamAΔ41 bölün. </li><li> Flash 193 ° K, sıvı azot ve mağaza donmuş </li></ol> 4. Temsilcisi Sonuçlar Bu protokol doğru yapıldığında bir yüksek derecede saflaştırılmış, boyut, homojen ve konsantre protein örneklerinin almalısınız. Bu protein örneklerinin yanı sıra kristalizasyon çalışmalarda, enzim kinetiği, afinitesi ve daha fazlası gibi biyokimyasal çalışmalar, daha sonra hazır. Arıtma protokolü ana adımlar temsilcisi sonuçlar burada nitelendirdi. Ni-NTA afinite sütun elüsyon profili SDS-PAGE analizi ~ 22kDa (Şekil 1) uygun MW mediğinin ifade protein üzerinde çok açığa çıkarmalıdır. Bu SDS-PAGE analizi de flow-through sınırsız proteinler herhangi bir protein, akış ve ultra santrifüj pelet örnek yıkama en az açığa çıkarmalıdır. Uygun protein büyük bantlar bu örnekler görünür yoksa bir yanlış tampon hazırlanması, hücre parçalama veya kültür oto-indüksiyon koşulları ve büyüme sorunları sorunları göz önünde bulundurmalısınız. İyon değişim kromatografisi nikel reçine (elektrostatik etkileşimleri veya histidin / olumsuz amino asitlerden zengin protein döngüler nedeniyle) ve kesilmemiş Onun Takip edilen arzu protein bağlı diğer E.coli proteinler arasında bizim arzu edilen proteini ayırmak için önceden şekillendirilmiş. Bu sütun artan NaCl konsantrasyonları altında pozitif yüklü reçine afinitesi göre proteinler ayırır. Yüksek olumsuz ücret proteini yüksek NaCl konsantrasyonları negatif yüklü olanları orta appose Zehir. Bu sütunun avantajları yüksek akış hızı ve bağlama kapasitesi vardır. Iyon değişimi kromatogram (Şekil 2) artan NaCl konsantrasyonu 3 proteinler nüfus arasında iyi bir ayırım ortaya koymaktadır. SDS-PAGE analizi, ileri arıtma adımları gerekli olup olmadığını arzu edilen bir ve değerlendirme tecrit, her nüfus MW belirlemek amacıyla ihtiyaç duyulmaktadır. Ikinci nüfus sığır trombin tarafından parçalanır ve üçüncü nüfusun SDS-PAGE düşük konsantrasyon nedeniyle mümkün değildir değildi MamAΔ41 + Onun Tag (~ 22kDa) ise ilk nüfus MamAΔ41 (~ 20 kDa). Proteinler doruklarına açıkça ayrılmış değilseniz bir yanlış tampon hazırlık düşünün veya NaCl degrade eğimi değiştirerek. Boyut dışlama kromatografisi Ni-NTA reçine bağlı ve iyon değişimi kromatografisi sırasında ayrılmış değil diğer E.coli hücre proteinleri bizim arzu protein arasındaki ayırmak için önceden şekillendirilmiş. Bu sütun, boylarına göre proteinler ayırır. Büyük proteinler karşı büyük hacimlerde yıkandı, küçük küçük elüsyon hacimleri Zehir. Sütun kromatogram (Şekil 3), iyi bir ana nüfus olarak arzu edilen bir protein ayırma ortaya koymaktadır. Nüfus uygun monomer boyutu ~ 20 kDa MW Zehir görünür. ~ 80 kDa bandı (Ni-NTA reçine bağlar E.coli tipik protein) varlığı sütun yükleme SDS-PAGE önce tespit edilir ve bu çalışması sırasında kaybolur. Bu ~ 80 kDa protein konsantrasyonu ile başlayan düşük olduğundan, nüfus, seyreltme ve SDS-PAGE analizi, arıtma etkinliğini belirlemek için gerekli nedeniyle kromatogram görünmüyor. Biz bir kesinlikle uygun MW saf protein varlığını belirlemek için SDS-PAGE ana zirve seyreltilmiş ve konsantre bir örnek yükleme önerilir. Bu aşamada proteinin saflaştırılmış ve homojenlik MALDI-TOF tarafından değerlendirmeye tabi tutulmalıdır. Bu analiz MW 10.176 Da (Şekil 4) Mw 20347 Da ve başka bir protein saptandı. ~ 10 kDa protein ~ 20 kDa protein ücret iki katına çıkar. His-Tag kaldırma sonra kalan 9 amino asitler ile MamAΔ41 tahmin MW 20.596,5 Da edildi. Elde edilen MALDI-TOF MW karşılaştırarak bunların dışında 248 Da bulundu. Bu benzemezliği MALDI-TOF ölçüm hataları ve / veya ilk metiyonin ortak bozulması ve ikinci glisin nedeniyle neden olabilir. Sonuç olarak, proteinin yüksek derecede saflaştırılmış ve daha fazla deneyler için kullanılabilir. <img src="/files/ftp_upload/1844/1844table.jpg" alt="Tablo 1" /> Tablo 1. Tampon formülasyonlar. <img src="/files/ftp_upload/1844/1844fig1.jpg" alt="Şekil 1" /> Şekil 1. Ni-NTA sütun arıtma Temsilcisi SDS-PAGE analizi, sağdan sola; P-ultra santrifüj pelet (3.11 protokol adım), W yıkama (3.12 protokol adım), U-sınırsız proteinleri (3.10 protokol adım), E - elüsyon samp beş şeritles (3.14 protokol adım), M-protein işaretleyici (sayı MW belirtiniz). Ok MamAΔ41 gösterir. <img src="/files/ftp_upload/1844/1844fig2.jpg" alt="Şekil 2" /> Şekil 2. Iyon değişim kromatografisi adım (a) İyon değişimi (MonoQ sütun) kromatogram Analizi; Mavi - toplanan fraksiyonları göstergesi emme 280nm, protein konsantrasyonu, Yeşil-NaCl konsantrasyonu, Kırmızı temsil eder. (B) iyon değişimi arıtma adım Temsilcisi SDS-PAGE analizi. Soldan sağa; M-protein işaretleyici (numaraları Mw göstermektedir), A6 - ilk zirve fraksiyonu, A8-ikinci zirve fraksiyonu. <img src="/files/ftp_upload/1844/1844fig3.jpg" alt="Şekil 3" /> Şekil 3. Boyut dışlama kromatografisi analizi (a) Boyutu hariç tutma (Superdex 200 sütun) kromatogram; Mavi - emilimi 280nm, protein konsantrasyonu, toplanan fraksiyonları Red göstergesi temsil eder . (B) boyut dışlama arıtma adım Temsilcisi SDS-PAGE analizi. Soldan sağa; M-protein işaretleyici (numaraları MW göstermektedir), PreI pre-enjekte örnek, ilk zirve toplanan A4-6 kombine fraksiyonu, A4-6D Seyreltilmiş fraksiyonu zirveden toplanır. <img src="/files/ftp_upload/1844/1844fig4.jpg" alt="Şekil 4" /> Şekil 4. Matriks yardımlı lazer desorpsiyon / iyonizasyon (MALDI-TOF) kütle spektrumu saflaştırılmış MamAΔ41. Matris Sinapic asit (SA). MamAΔ41 de gösterildiği gibi, 20.347 Da gösterilen 10.176 Da MamAΔ41 iki katına ücret türler.

<ol>
	<li>Faivre, D., Schuler, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetotactic+Bacteria+and+Magnetosomes.">Magnetotactic Bacteria and Magnetosomes.</a> Chem Rev. 108, 4875-4898 (2008).</li><li>D'Andrea, L. D., Regan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TPR+proteins:+the+versatile+helix.">TPR proteins: the versatile helix.</a> Trends Biochem Sci. 28, 655-662 (2003).</li><li>Young, J. C., Barral, J. M., Hartl, U. l. r. i. c. h., F, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=More+than+folding:+localized+functions+of+cytosolic+chaperones.">More than folding: localized functions of cytosolic chaperones.</a> Trends Biochem Sci. 28, 541-547 (2003).</li><li>Brocard, C., Hartig, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peroxisome+targeting+signal+1:+is+it+really+a+simple+tripeptide?.">Peroxisome targeting signal 1: is it really a simple tripeptide?.</a> Biochim Biophys Acta. 1763, 1565-1573 (2006).</li><li>Fransen, M., Amery, L., Hartig, A., Brees, C., Rabijns, A., Mannaerts, G. P., Van Veldhoven, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+PTS1-+and+Rab8b-binding+properties+of+Pex5p+and+Pex5Rp/TRIP8b.">Comparison of the PTS1- and Rab8b-binding properties of Pex5p and Pex5Rp/TRIP8b.</a> Biochim Biophys Acta. 1783, 864-873 (2008).</li><li>Baker, M. J., Frazier, A. E., Gulbis, J. M., Ryan, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+protein-import+machinery:+correlating+structure+with+function.">Mitochondrial protein-import machinery: correlating structure with function.</a> Trends Cell Biol. 17, 456-464 (2007).</li><li>Mirus, O., Bionda, T., von Haeseler, A., Schleiff, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionarily+evolved+discriminators+in+the+3-TPR+domain+of+the+Toc64+family+involved+in+protein+translocation+at+the+outer+membrane+of+chloroplasts+and+mitochondria.">Evolutionarily evolved discriminators in the 3-TPR domain of the Toc64 family involved in protein translocation at the outer membrane of chloroplasts and mitochondria.</a> J Mol Model. 15, 971-982 (2009).</li><li>Gatsos, X., Perry, A. J., Anwari, K., Dolezal, P., Wolynec, P. P., Likic, V. A., Purcell, A. W., Buchanan, S. K., Lithgow, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+secretion+and+outer+membrane+assembly+in+Alphaproteobacteria.">Protein secretion and outer membrane assembly in Alphaproteobacteria.</a> FEMS Microbiol Rev. 32, 995-1009 (2008).</li><li>Tiwari, D., Singh, R. K., Goswami, K., Verma, S. K., Prakash, B., Nandicoori, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Key+residues+in+Mycobacterium+tuberculosis+protein+kinase+G+play+a+role+in+regulating+kinase+activity+and+survival+in+the+host.">Key residues in Mycobacterium tuberculosis protein kinase G play a role in regulating kinase activity and survival in the host.</a> J Biol Chem. 284, 27467-27479 (2009).</li><li>Edqvist, P. J., Broms, J. E., Betts, H. J., Forsberg, A., Pallen, M. J., Francis, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetratricopeptide+repeats+in+the+type+III+secretion+chaperone,+LcrH:+their+role+in+substrate+binding+and+secretion.">Tetratricopeptide repeats in the type III secretion chaperone, LcrH: their role in substrate binding and secretion.</a> Mol Microbiol. 59, 31-44 (2006).</li><li>Grunberg, K., Muller, E. C., Otto, A., Reszka, R., Linder, D., Kube, M., Reinhardt, R., Schuler, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+proteomic+analysis+of+the+magnetosome+membrane+in+Magnetospirillum+gryphiswaldense.">Biochemical and proteomic analysis of the magnetosome membrane in Magnetospirillum gryphiswaldense.</a> Appl Environ Microbiol. 70, 1040-1050 (2004).</li><li>Komeili, A., Vali, H., Beveridge, T. J., Newman, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetosome+vesicles+are+present+before+magnetite+formation,+and+MamA+is+required+for+their+activation.">Magnetosome vesicles are present before magnetite formation, and MamA is required for their activation.</a> Proc Natl Acad Sci USA. 101, 3839-3843 (2004).</li><li>Okuda, Y., Fukumori, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+characterization+of+a+magnetosome-associated+protein,+TPR-containing+MAM22,+in+Escherichia+coli.">Expression and characterization of a magnetosome-associated protein, TPR-containing MAM22, in Escherichia coli.</a> FEBS Lett. 491, 169-173 (2001).</li><li>Taoka, A., Asada, R., Sasaki, H., Anzawa, K., Wu, L. F., Fukumori, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+localizations+of+Mam22+and+Mam12+in+the+magnetosomes+of+Magnetospirillum+magnetotacticum.">Spatial localizations of Mam22 and Mam12 in the magnetosomes of Magnetospirillum magnetotacticum.</a> J Bacteriol. 188, 3805-3812 (2006).</li><li>Studier, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+production+by+auto-induction+in+high+density+shaking+cultures.">Protein production by auto-induction in high density shaking cultures.</a> Protein Expression and Purification. 41, 207-234 (2005).</li></ol>

Proteinlerin saflaştırılması, herhangi bir biyokimyasal proteinler ya da yapısal çalışmalar içinde önemli bir adımdır. Her protein kendi davranışları ile benzersiz olduğu için, onun özelliklerini tanımlamak ve buna göre arıtma değiştirmek gerekiyor. Protein hedef biyoinformatik araçlar kullanarak arıtma doğru bir ilk adım olarak analiz edilmelidir. Onlar, hedef iso-elektrik nokta hesaplamak için çevre ve özel iyonları / ligandlar için ihtiyacı azaltıcı / oksitleyici ihtiyacı değerlen...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>French Press</td>
<td>Equipment</td>
<td>Thermo scientific</td>
<td>FA-078A</td>
<td>&nbsp;</td>
<tr>
<td>Pressure cell</td>
<td>Equipment</td>
<td>Thermo scientific</td>
<td>FA-032</td>
<td>&nbsp;</td>
<tr>
<td>Ultra-centrifuge</td>
<td>Equipment</td>
<td>Sorvall</td>
<td>Discovery 90SE</td>
<td>&nbsp;</td>
<tr>
<td>Rottor</td>
<td>Equipment</td>
<td>Beckman</td>
<td>Ti60</td>
<td>&nbsp;</td>
<tr>
<td>Ultra-centrifuge tubes; PC-Bottle+Cap Assay 26.3ml</td>
<td>Equipment</td>
<td>Beckman</td>
<td>BC-355618</td>
<td>&nbsp;</td>
<tr>
<td>2.5cm diameter, Glass Econo-Column Chromatography Columns</td>
<td>Equipment</td>
<td>BioRad</td>
<td>737-2521</td>
<td>&nbsp;</td>
<tr>
<td>Ni-NTA His Bind resin</td>
<td>Equipment</td>
<td>Novagen</td>
<td>M0063428</td>
<td>&nbsp;</td>
<tr>
<td>Spectrophotometer</td>
<td>Equipment</td>
<td>Amersham Biosiences</td>
<td>Ultraspec 2100 pro</td>
<td>&nbsp;</td>
<tr>
<td>Quartz cuvette</td>
<td>Equipment</td>
<td>Hellma</td>
<td>104-QS</td>
<td>&nbsp;</td>
<tr>
<td>Fast Performance Liquid Chromatography- AKTA purifier 10</td>
<td>Equipment</td>
<td>GE Healthcare Biosciences</td>
<td>28-4062-64</td>
<td>&nbsp;</td>
<tr>
<td>Ion exchange column &#x2013; MonoQ 4.6/100 PE</td>
<td>Equipment</td>
<td>GE Healthcare Biosciences</td>
<td>10025543</td>
<td>&nbsp;</td>
<tr>
<td>Size exclusion pre-packed column-HiLoad 26/60 Superdex 200</td>
<td>Equipment</td>
<td>GE Healthcare Biosciences</td>
<td>17-1071-01</td>
<td>&nbsp;</td>
<tr>
<td>Centricon - Vivaspin15 &#x2013; 10,000 MWCO</td>
<td>Equipment</td>
<td>Sartorius Stedim Biotech GmbH</td>
<td>VS1501</td>
<td>&nbsp;</td>
<tr>
<td>Table centrifuge</td>
<td>Equipment</td>
<td>Thermo scientific</td>
<td>IEC CL30R</td>
<td>&nbsp;</td>
<tr>
<td>MALDI-TOF</td>
<td>Equipment</td>
<td>Bruker Daltonics</td>
<td>Reflex IV</td>
<td>&nbsp;</td>
<tr>
<td>Tris-HCl (hydrotymethyl) aminomethane</td>
<td>Reagent</td>
<td>BioLab</td>
<td>20092391</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Chloride</td>
<td>Reagent</td>
<td>FRUTROM</td>
<td>235553470</td>
<td>&nbsp;</td>
<tr>
<td>Imidazole</td>
<td>Reagent</td>
<td>Alfa Aesar</td>
<td>288-32-4</td>
<td>&nbsp;</td>
<tr>
<td>EDTA free protease inhibitors cocktail</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P-8849</td>
<td>&nbsp;</td>
<tr>
<td>Dnase I (Deoxyribonuclease I)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>DN-25</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Thrombin</td>
<td>Reagent</td>
<td>Fisher BioReagents</td>
<td>BP25432</td>
<td>&nbsp;</td>
<tr>
<td>Glycine</td>
<td>Reagent</td>
<td>BioLab</td>
<td>07132391</td>
<td>&nbsp;</td>
<tr>
<td>Soudim Dodecyl Sulfate (SDS)</td>
<td>Reagent</td>
<td>BioLab</td>
<td>19822391</td>
<td>&nbsp;</td>
<tr>
<td>Beta-mercaptoethanol</td>
<td>Reagent</td>
<td>Sigma</td>
<td>M-3148</td>
<td>&nbsp;</td>
<tr>
<td>InstantBlue</td>
<td>Reagent</td>
<td>Expedeon</td>
<td>1SB01L</td>
<td>&nbsp;</td>
<tr>
<td>PageRuler Prestained Protein Ladder</td>
<td>Reagent</td>
<td>Fermentas</td>
<td>SM0671</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

purification of the m. magneticum strain amb-1 magnetosome associated protein mama&delta;41

Magnetotactic bakteri jeomanyetik alanlar boyunca kendi yolunuzu mümkün Sudaki mikroorganizmaların çeşitli bir grup oluşturmaktadır. Bu davranış uygun ortamlar için arama yardımcı olduğuna inanılıyor<em&gt; (1)</em&gt;. Bu yetenek magnetosome öngördüğü, lipid lineer bir zincir montaj oluşan bir hücre içi organel manyetit veya greigite ~ 50-nm kristal biomineralize ve içine her mümkün vezikül. Fonksiyonel veziküllerin oluşumu için gerekli olduğu gösterilmiştir magnetosome bir ilke bileşeni Mama. Mama en karakterize magnetosome ilişkili proteinlerin biridir oldukça bol magnetosome ilişkili protein<em&gt; In vivo</em&gt;<em&gt; (2-6)</em&gt;. Bu makale, Mama arıtma odaklanır çalışılan rağmen<em&gt; In vivo</em&gt; Net fonksiyonel veya yapısal ayrıntılar için tespit edilmiştir. Biyoinformatik analizi Mama, protein içeren bir tetra-tricopeptide tekrar (TPR) olduğunu ileri sürdü. TPR proteinlerin geniş bir yelpazede büyük bir kat ya da yapıcı bir parçası olarak bulunan bir yapısal motif, protein-protein etkileşimleri ve aracılık çok protein kompleksleri için bir şablon olarak hizmet vermektedir<em&gt; (7)</em&gt;. TPRs ökaryotik hücre organel süreçleri ve birçok bakteriyel yollar çok önemli görevlerde yer<em&gt; (8-14).</emMama anlamak&gt; için, protein içeren eşsiz bir TPR, bir ilk adım olarak yüksek derecede saflaştırılmış protein gereklidir. Bu makalede, istikrarlı bir Mama silme mutant (MamAΔ41) arıtma protokolü mevcut<em&gt; M. magneticum</em&gt; AMB-1.

Watch this Scientific Journal Video about Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamAÎ”41 at JoVE.com

Purification of the &lt;em&gt;M. magneticum&lt;/em&gt; Strain AMB-1 Magnetosome Associated Protein MamA&amp;Delta;41

Mama magnetosome aktivasyonu dahil olmak gösterildi eşsiz bir Magnetosome ilişkili protein. Burada Mama silme mutant arıtma protokolü (MamAΔ41) mevcut<em&gt; M. magneticum</em&gt; AMB-1.

Saflaştırılması M. magneticum Deformasyon AMB-1 Magnetosome İlişkili Protein MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

purification-m-magneticum-strain-amb-1-magnetosome-associated-protein

Research

JoVE Journal

Biology

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

13.4K Görüntüleme.  Ben-Gurion University. Mama magnetosome aktivasyonu dahil olmak gösterildi eşsiz bir Magnetosome ilişkili protein. Burada Mama silme mutant arıtma protokolü (MamAΔ41) mevcut M. magneticum AMB-1.

Video: Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

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

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Sıvı Faz Kurtarma Moleküler Ağırlık Tabanlı Fraksiyonu GELFREE 8100 Fraksiyonu Sisteminin Kullanılması

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated anti-cancer drugs or antibiotics, is a current serious problem in public health. This multidrug resistance is largely due to energy-dependent drug efflux pumps. The pumps expel anti-cancer drugs or antibiotics into the external medium, lowering their intracellular concentration below a toxic threshold. We are studying multidrug resistance in Pseudomonas aeruginosa, an opportunistic bacterial pathogen that causes infections in patients with many types of injuries or illness, for example, burns or cystic fibrosis, and also in immuno-compromised cancer, dialysis, and transplantation patients. The major MDR efflux pumps in P. aeruginosa are tripartite complexes comprised of an inner membrane proton-drug antiporter (RND), an outer membrane channel (OMF), and a periplasmic linker protein (MFP) 1-8. The RND and OMF proteins are transmembrane proteins. Transmembrane proteins make up more than 30% of all proteins and are 65% of current drug targets. The hydrophobic transmembrane domains make the proteins insoluble in aqueous buffer. Before a transmembrane protein can be purified, it is necessary to find buffer conditions containing a mild detergent that enable the protein to be solubilized as a protein detergent complex (PDC) 9-11. In this example, we use an RND protein, the P. aeruginosa MexB transmembrane transporter, to demonstrate how to express a recombinant form of a transmembrane protein, solubilize it using detergents, and then purify the protein detergent complexes. This general method can be applied to the expression, purification, and solubilization of many other recombinantly expressed membrane proteins. The protein detergent complexes can later be used for biochemical or biophysical characterization including X-ray crystal structure determination or crosslinking studies.

In this protocol we demonstrate the expression, solubilization, and purification of a recombinantly expressed membrane protein, MexB, as a soluble protein detergent complex. MexB is a multidrug resistance membrane transporter from the opportunistic bacterial pathogen Pseudomonas aeruginosa.

İfade, Deterjan çözünürleştirme ve Membran Transporter, MexB ilaca Direnç Protein Saflaştırılması

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated ...

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

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 ...

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 ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

Yılında Kistik Fibrozis Transmembran İletkenlik Regülatörü Protein İfadesi ve Arıtma Saccharomyces cerevisiae

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Fonksiyon Çalışma, Etkileşimler, ve Post-translasyonel değişiklikler için tomurcuklanan Maya Protein Ekstraksiyonu ve Saflaştırılması

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

Plant protein kompleksleri, çevirim sonrası değişikliklerin belirlenmesi

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;