Bu çalışma NIH (5T32GM008275 22) ve Amerikan Kalp Derneği (EAS) predoctoral dostluk bir hibe ile desteklenen; Kimya-Biyoloji Arabirimi NIH (2T32GM071339-06A1) (MED) Bursu; ve hibe NIH (1DP2OD002177-01 ve NS067354-0110), Ellison Tıp Vakfı ve Bill ve Melinda Gates Vakfı (JS).

1. Hsp104, Açıklanması <ol><li> Plazmid E. arıtma için istihdam coli, pPROEX-HTB-Hsp104, trc organizatörü 26 indüklenebilir kontrol altında Hsp104 açık okuma çerçevesi içerir. Plazmid TEV proteaz bölünme tarafından kaldırılabilir bir N-terminal Onun 6-etiket Hsp104 üretir. Kodon optimize edilmiş E. içine pPROEX-HTB-Hsp104 Dönüşümü tipik bir bakteriyel dönüşüm prosedürü kullanarak coli BL21-CodonPlus RIL hücreleri (Stratagene, Agilent Technologies) (örneğin, üreticinin talimatlarına göre). Kodon optimize edilmiş E. kullanmak önemlidir. Hsp104 alışılmadık bir kodon önyargı çünkü coli süzün. </li><li> 100 mL 2XYT kültür (USB. Tarif: 16g / L kazein pepton, 10 g / L maya özü, 5 g / L NaCl, pH 7.0) İnokülasyon 100μg/mL ampisilin ve taze transformants 34μg/mL kloramfenikol ile desteklenmiş ve 37 o gecede büyümek ° 200rpm az sallayarak C. </li><li> 6 X 2L şişeler 100μg/mL ampisilin ve 34μg/mL kloramfenikol 1L 2XYT içine 10 ml gecelik kültür İnokülasyon. 37 ° C kadar OD 600 = 0.4-0.6 250rpm az çalkalayınız. Sallayarak durdurun ve 15 sıcaklığını düşürmek ° C 15 ° C&#39;ye ulaşıncaya kadar hücrelerin inkübatör 30dk için unagitated denge sağlaması için izin verin Bir kez 15 ° C 1mm bir final konsantrasyon IPTG ekleyerek protein ekspresyonu neden ulaşılır. (12-16 saat) bir gecede 250rpm sallayarak sürdürün. </li></ol> 2. Hücre Hasat ve Lizis <ol><li> 4 20dk için 4.000 rpm santrifüj Hasat hücreleri (bir precooled rotor) ° C (Sorvall RC 3BP + santrifüj kullanın). Hsp104 aktivitesi azaldı çünkü sonraki adımları derhal yapılmalıdır E. coli hücrelerinde dondurulur. Ayrıca, sonraki tüm adımlar buz veya 4 ° C&#39;de yapılmalıdır </li><li> Prechilled yeniden süspanse hücre pelletleri (buz) 10ml lizis tamponu (40mm Hepes-KOH pH 7.4, 500mm KCl, 20mm MgCl 2,% 2,5 (w / v) gliserol, 20mm imidazol, 5μM pepstatin, tam proteaz inhibitörü kokteyl (1 EDTA tablet/50mL-ücretsiz), 2mm β-mercaptoethanol). </li><li> Buzlu suya dalmış bir ısı değiştirici kullanan bir Fransız basını (Emulsiflex) Homojenizatör hücreleri Lyse. Kullanmadan önce, tüm hücre kümeleri çözündüğünü sahip olduğunuzdan emin olun. Lizis tamponu ile homojenizatör değişikti sonra, iki geçiş 15,000-18,000 psi basıncı ile silindir üzerinden tam lizis için yeterli. Fransız basını, mevcut değilse, sonication takip lizozim ile inkübasyon (tartışma) kullanılabilir. SDS-PAGE analizi (1μL) (Şekil Lizat 2) parçalanmış hücreleri küçük bir örnek. </li><li> 4 20dk 16.000 rpm&#39;de santrifüj hücre enkaz kaldırma ° C (+ centifuge bir Sorvall RC5C kullanın). Bir sonraki adım için supernatant saklayın ve SDS-PAGE analizi için süpernatant küçük bir örneği (1μL) tasarruf (Ni Yük Şekil 2). </li></ol> 3. Hsp104 Arıtma <ol><li> Lizis tamponu dengelenmiş Ni-sepharose boncuk (GE)% 50 bulamaç 12ml (1L hücre başına 2mL Ni-sepharose boncuk) ile 2,4 adım supernatant karıştırın. </li><li> 4 az 3 saat yavaş örnek Döndür ° C Ni-sepharose eşit kapanmıştır ve Köpüklenmemesine minimize kaldığı gibi. Kısa kuluçka süreleri mümkündür, ancak verim düşecektir. 4 ° C proteaz inhibitörlerinin varlığında, küçük bir bozulma meydana gelir ve Ni-sepharose 3 saatlik bir inkübasyon süresi, nihai ürünün etkinliği düşmediğinden. 2 dakika süreyle santrifüj Ni-sepharose toplayın, 4 ° C &#39;de 2.000 tane rpm (Eppendorf santrifüj 5810R). SDS-PAGE analizi (1μL) kaydedin ve süpernatant bir küçük örnek (Şekil sayesinde Ni Akış (FT) 2) geri kalanını atın. </li><li> Yıkama tamponu (40mm Hepes-KOH pH 7.4, 150mm KCl, 20mm MgCl 2,% 2.5 (w / v) gliserol, 20mm imidazol, 2mm β-mercaptoethanol), 5 sütun hacimleri 25 sütun hacimleri ile alınan Ni-sepharose yıkayın Hsp104 elektrostatik bağlı kirleticiler ve yıkama tamponu 25 sütun hacimleri kaldırmak için 1M KCl ile tampon yıkayın. 2 dakika süreyle santrifüj her yıkamadan sonra Ni-sepharose toplayın, 4 ° C &#39;de 2.000 tane rpm (Eppendorf santrifüj 5810R). Her yıkama ve santrifüj çevriminden sonra, aspirasyon tampon kaldırmak. </li><li> Hsp104, mix Ni-sepharose 1mL elüsyon 1mL Ni-sepharose başına tamponu (40mm Hepes-KOH pH 7.4, 150mm KCl, 20mm MgCl 2,% 2.5 (w / v) gliserol, 350mm imidazol, 2mm β-mercaptoethanol) ve Zehir 4 ° C&#39;de 20 dakika çevirin. Yüksek imidazol konsantrasyon Ni boncuk 6 etkileşimi bozar. 4 az 2 dakika süreyle santrifüj boş 1.2 mL dönüş sütun (Bio-Rad) ° C 2.000 rpm (Eppendorf 5810R santrifüj) Ni-sepharose çıkarın. (Şekil Ni eluat 2) SDS-PAGE analizi için eluat, 1μL kaydedin. ~ 15mg, Hsp104 hücrelerin her litre için, bu aşamada elde edilir. </li><li> MWCO 30.000 Amicon Ultra (Mil kullanarak içine Tampon Tampon Q (20mm Tris-HCl, pH 8, 0.5mm EDTA, 5mM MgCl 2, 50mm NaCl) döviz eluatlipore), 15 mL santrifüj konsantratör birimleri. Birincisi, daha sonra 10 kat protein sulandırmak için Tampon Q 14.5mL eklemek ~ 1.5mL protein konsantresi. Tampon değişimi için 3 kez tekrarlayın. Artan pH ve düşük tuz Hsp104 yüksek bir negatif yük sahip olmasını sağlar. </li><li> Buffer S dengelenmiş Kaynak S 6mL sütun (GE) ile anyon değişim kromatografisi yoluyla Hsp104 arındırın. Enjeksiyondan önce, protein, düşük protein bağlayıcı Millex GP PES Membran 0.22μM şırınga filtresi (Millipore) süzülür. Biz 1mL/min bir akış oranı istihdam etmektedir. 1 sütun hacmi ile% 20 Buffer zayıf bağlayıcı proteinlerin uzaklıkta yıkayın S + (20mm Tris-HCl, pH 8, 0.5mm EDTA, 5mM MgCl 2, 1M NaCl). 5 sütun hacimleri üzerinden doğrusal bir degrade Zehir Hsp104 (20% -50% Tampon S +) (Şekil 3). 1ml kesirler toplayın. Hsp104 ve türevlerini en tipik ~ 400mm NaCl (31mS/cm) Zehir. SDS-PAGE analizi (Şekil 3, içerlek) pik fraksiyonları (1μL) örnekleri kaydedin. </li><li> Amicon santrifüj konsantratör kullanarak, O&#39;nun 6-tag kaldırmak için, döviz protein olarak bölünme tampon (20mm Hepes-KOH pH 7.4, 140 KCl ve 10mM MgCl 2) (bkz: adım 3.5) yukarıda tarif. Üreticinin talimatlarına göre proTEV proteaz (Promega) veya AcTEV proteaz (Invitrogen) kullanın. Hsp104 için, biz Hsp104 tam bölünme için gerekli olan: TEV proteaz yüksek oranları bu bulduk. Biz 12μg Hsp104 başına 1 proteaz biriminin bir oran kullanın. Dilinim 2-4 saat süreyle, 16 saat inkübasyon 4 ° C 30 ° C&#39;de olmalıdır Final Hsp104 konsantrasyonu 20-75μM monomer arasında olmalıdır. Ni-sepharose (GE) ile kalan His6 etiketli Hsp104 ve TEV proteaz fazla bölünme tepki Hsp104 miktarı (boncuklar paketlenmiş reçine ml başına protein 15mg bağlayabilirsiniz varsayalım) Ni-sepharose ekleyerek tüketir. 4 2min için 2,000 rpm&#39;de santrifüj boş dönmeye sütun (Bio-Rad) ile boncuk kaldırın ° C Bölünme ve uncleaved protein (Şekil 4) kaldırılmasını sağlamak için SDS-PAGE analizi için bölünme (1μL) öncesi ve sonrası numunelerini. </li><li> MWCO 30.000 Amicon Ultra (Millipore) kullanarak adım 3.5 &#39;te açıklandığı gibi boyut dışlama tampon (20mm Hepes-KOH pH 7.4, 140 KCl, 10mM MgCl 2, 0.1mm EDTA, 1mm DTT) içine Alım Satım. </li><li> Ayrıca boyut dışlama kromatografisi ile Hsp104 arındırmak. Boyut dışlama tampon dengelenmiş Superose 6 veya Superdex 200 sütun (GE) (Şekil 5) kullanın. Enjeksiyondan önce, protein, düşük protein bağlayıcı Millex GP PES Membran 0.22μM şırınga filtresi (Millipore) süzülür. Daha az protein 10mg, 10/300 boyutu sütun (24mL) ve 0.5 ml kesirler toplanır. 10mg daha büyük numuneler için, 12/60 boyutu kolon (120ml) ve 1 ml kesirler toplanan. Akış oranları ve yüklenecek örnek hacmi için üreticinin talimatlarına bakın. Saflaştırma sonrasında, Hsp104 kesirler Şekil toplanmış. 5 ve Amicon Santrifüj Konsantratörü Birimleri (Millipore) yoğunlaşmıştır. Aşağıda açıklandığı gibi Hsp104 saklanır. Tam uzunlukta Hsp104 saflaştırılmış nihai verim kültürünün başlangıç ​​~ 1-3mg litre Hsp104 bozunma ürünleri ve kirleticiler ayıran malzeme kaybı nedeniyle. </li></ol> 4. Hsp104 Disaggregase aktivitesi <ol><li> Arıtma sonra, bir ayrıştırılmasıyla tayininde Hsp104 etkinliğini değerlendirmek için iyi bir uygulamadır. Genellikle, bir lusiferaz reaktivasyonu tahlil 2 (Şekil 6) kullanırlar. Bu testte, HSP70 şaperon sistemi (HSP70 ve Hsp40) disaggregates üre-denatüre ateşböceği lusiferaz agrega 2 ile birlikte Hsp104. Çözünür lusiferaz oxyluciferin, ışık bültenleri bir tepki luciferase oksidasyonunu katalizler. Izleme lüminesans, göreceli reaktivasyonu, ve bu nedenle ayrıştırılmasıyla, lusiferaz tespit edilebilir. Hsp104 HSP70 co-şaperon sistemi ile sinerjik işbirliği mümkün olmalıdır. Kurtarıldı lüminesans miktarı tam HSP70 bağlıdır: Hsp40 çifti kullanılmaktadır. Biz rutin Hsp72 ve Hsp40 (Tahlil Tasarımlar) kullanırlar. Yalnız Hsp40 (Şekil 6): En azından, aktif Hsp104 lusiferaz reaktivasyonu bir Hsp72 ile reaktivasyonuna karşılaştırıldığında 5 kat artış üretmek gerekir. Faaliyet bu seviyeye ulaşmak için başarısız Hsp104 hazırlıkları atılır. </li></ol> 5. Hsp104 Depolama <ol><li> Kısa süreli depolama için, Hsp104 4 ° C boyut-dışlanma tampon tutulabilir. Ancak, aktivite, 4 4 ° C ve keskin 1 hafta sonra 2-3 gün sonra azalacak ° C Ayrıştırılmasıyla testlerde Hsp104 arıtma sonra en kısa sürede kullanılması gerektiğini öneriyoruz. İdeal olarak, Hsp104 amiloid ayrıştırılmasıyla deneyleri için hemen kullanılabilir olmalıdır. </li><li> Protein uzun vadeli depolanması gerekiyorsa Hsp104 depolama tampon (20mm Hepes-KOH pH 7.4, 140 KCl, 10mM MgCl 2, 0.1mm EDTA, 1mm DTT,% 10 gliserol (w / v)) değiştirilir. 100μl alikotları sıvı nitrojen içinde dondurulmuş ve -80 ° C&#39;de saklanan ek bileşenini Donma-çözülme çevrimleri Hsp104 aktivite önemli ölçüde azaltmakve bundan kaçınılmalıdır. Biz yavaş yavaş buz üzerinde Hsp104 çözülme tavsiye ederiz. Amiloid-disaggregase faaliyet -80 az 1 ay sonra keskin bir düşüş olacak ° C </li></ol> 6. Temsilcisi Sonuçlar ve Şekiller: <img src="/files/ftp_upload/3190/3190fig1.jpg" alt="Şekil 1"/> Şekil 1. Hsp104 Bifonksiyonel disaggregase. Düzensiz agrega (solda gösterilen) Disagregasyon HSP70 şaperon sistemi (HSP70 ve Hsp40) 2 işbirliğini gerektirir. Hsp104 remodels in vitro HSP70 ve Hsp40 yardımı olmaksızın amiloid agrega (sağda gösterildiği gibi) emretti, ama HSP70 ve Hsp40 amiloid 26,28 karşı Hsp104 etkinliği artırabilir. Ayrıştırılmasıyla teşvik etmek için merkezi bir kanal aracılığıyla substrat translokasyon Hsp104 çiftler ATP hidrolizi toplu yapılar, her iki tip. Tirozin taşıyan gözenek döngüler, merkezi kanal 49-52 yoluyla servis substrat meşgul . <img src="/files/ftp_upload/3190/3190fig2.jpg" alt="Şekil 2"/> Şekil 2. Ni-sepharose yakınlık arıtma adım SDS-PAGE analizi. Lizat, Ni Yük, Ni FT ve Ni eluat örnekleri% 4-20 Tris-HCl 1.0mm Kriter jel (Bio-Rad) ve Coomassie vitray kullanarak SDS-PAGE ile fraksiyone . Tüm Hsp104 Ni-sepharose bağlamak mümkün olduğunu unutmayın. Broad Range molekül ağırlığı belirteçleri (Bio-Rad) (şeritli solda) gösterilmiştir. <img src="/files/ftp_upload/3190/3190fig3.jpg" alt="Şekil 3"/> Şekil 3. Ni-sepharose eluat Kaynak S arıtma Mavi iz 280nm ve yeşil iz absorbans temsil% Tampon S + (maksimum% 50) temsil eder. % 20 oranında elutes ilk zirve, Q + kirleri, bozunma ürünleri ve yanlış katlanmış Hsp104 içerir. Ikinci ve önemli bir zirve düzgün katlanmış ve aktif Hsp104 içerir. Akış hızı 1ml/min idi. % 20-50 Q + Gradient 30 dakika ya da 5 sütun hacimleri. Ankastre: tepe fraksiyonları% 4-20 Tris-HCl 1.0mm Kriter jel (Bio-Rad) ve Coomassie vitray kullanarak SDS-PAGE analizi ile çözümlenir. Broad Range molekül ağırlığı belirteçleri (Bio-Rad) (solda) gösterilmiştir. <img src="/files/ftp_upload/3190/3190fig4.jpg" alt="Şekil 4"/> Şekil 4. ProTEV proteaz bölünme adım SDS-PAGE analizi, 6-Hsp104 Kaynak S arıtma, 30 ° C daha sonra 4 ° C ve 16 saat 4 saat proTEV proteaz ile tedavi edildi Örnekler% 4-20 Tris-HCl 1.0mm Kriter jel (Bio-Rad) ve Coomassie vitray kullanarak SDS-PAGE ile fraksiyone. ProTEV bölünmüş Hsp104 daha hızlı göç olduğunu unutmayın. Adım 3.7 &#39;de açıklandığı gibi TEV proteaz ve uncleaved Hsp104 Ni-sepharose tükenmiş. Broad Range molekül ağırlığı belirteçleri (Bio-Rad) (solda) gösterilmiştir. <img src="/files/ftp_upload/3190/3190fig5.jpg" alt="Şekil 5"/> Şekil 5. Hsp104 boyutu dışlanma arıtma Hsp104 Superose 6 jel filtrasyon sütun (10/300, GE) ile saflaştırılmış kesildiler. Debi = 0.4ml/min. Kesik çizgiler arasında zirve toplanmış fraksiyonları temsil eder. Ankastre: tepe fraksiyonları% 4-20 Tris-HCl 1.0mm Kriter jel (Bio-Rad) ve Coomassie vitray kullanarak SDS-PAGE analizi ile çözümlenir. Broad Range molekül ağırlığı belirteçleri (Bio-Rad) (solda) gösterilmiştir. <img src="/files/ftp_upload/3190/3190fig6.jpg" alt="Şekil 6"/> Şekil 6. Lusiferaz reaktivasyonu tahlil Denatüre ateşböceği lusiferaz agrega (50 nm) Hsp72 ve Hsp40 (1 mikrona kadar) (Testi Tasarımlar hem de), Hsp104 (6μM monomer) veya Hsp104, Hsp72 ve 90 dk için Hsp40 ya da 25 ° C&#39;de inkübe edildi. Denatüre lusiferaz sadece Hsp104 ve Hsp40/Hsp72 varlığı tamamen yeniden. Kurtarılan lüminesans Sonsuz M1000 plaka okuyucu (Tecan) ölçülür. Değerler anlamına gelir ± SEM (n = 3) temsil eder.

Çıkar çatışması ilan etti.

Zaman Çizelgesi: maksimum Hsp104 aktivite için tüm arıtma düzeni mümkün olduğunca hızlı bir şekilde tamamlanmış olması tavsiye edilir . Ancak, arıtma adımları her zaman pratik olmayabilir zorlu bir program yapar. Saflaştırma adımları en kısa sürede yapılması durumunda, 30 inkübasyon 2-4 saat gece ifade sonundan itibaren zaman ° C TEV proteaz ile yaklaşık 9-11 saattir. Duraklatmak için potansiyel bir yer TEV bölünme adım takip ediyor. Kesinlikle gerekli ise, Hsp104 ek bileşenini (...

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th></tr><tr><td> BL21-CodonPlus-RIL Yetkili Hücreler </td><td> Stratagene, Agilent Technologies </td><td> 230255 </td></tr><tr><td> 2XYT suyu </td><td> USB </td><td> 75864 </td></tr><tr><td> Eksiksiz, minivan, EDTA içermeyen proteaz inhibitörü tabletler </td><td> Roche </td><td> 1836170 </td></tr><tr><td> Pepstatin A </td><td> Sigma </td><td> P4265 </td></tr><tr><td> Ni-sepharose 6 Hızlı Akış </td><td> GE Healthcare </td><td> 17-5318-02 </td></tr><tr><td> Amicon Ultra-15 santrifüj filtre üniteleri (MWCO 30.000) </td><td> Millipore </td><td> UFC903008 </td></tr><tr><td> Kaynak Q - 6ml sütun </td><td> GE Healthcare </td><td> 17-1179-01 </td></tr><tr><td> proTEV Proteaz </td><td> Promega </td><td> V6052 </td></tr><tr><td> AcTEV Proteaz </td><td> Invitrogen </td><td> 12575015 </td></tr><tr><td> Superose 6 10/300 GL </td><td> GE Healthcare </td><td> 17-5172-01 </td></tr><tr><td> Hsp40 </td><td> Testi Tasarımlar </td><td> SPP-400 </td></tr><tr><td> Hsp72 </td><td> Testi Tasarımlar </td><td> ADI-NSP-555 </td></tr></tbody></table>

purification of hsp104, a protein disaggregase

Hsp104 maya hexameric bir AAA + protein 1, 2-10 protein ayrıştırılmasıyla çiftler ATP hidrolizi (Şekil 1). Bu etkinlik iki anahtar seçici avantaj kazandırır. Birincisi, Hsp104 tarafından düzensiz agrega Yeniden doğallaştırmanın ısı şok 3,5,11,12 dahil olmak üzere, çeşitli protein misfolding gerilmeler sonra maya hayatta kalma güçlendirir. İkincisi, Hsp104 çapraz beta amiloid fibriller itilme, sayısız yarar sağlayan bir rezervuar olarak prion (enfeksiyöz amiloidleri) yararlanmak ve 13-22 fenotipik varyasyon kalıtsal maya sağlar . Enteresandır ki, Hsp104 doğrudan maya prion proteinlerin Sup35 ve Ure2 23-30 oluşan olanlar da dahil olmak üzere preamyloid oligomerler ve amiloid fibriller remodels. Bu amiloid remodeling işlevselliği maya Hsp104 özel bir yönüdür. E. coli orthologue, ClpB, yeni model preamyloid oligomerler veya amiloid fibriller 26,31,32 başarısız olur. Hsp104 orthologues hayvanlar, perplexingly dışında yaşamın tüm krallıkları bulundu. Gerçekten de, hayvan hücrelerinde herhangi bir enzim sistemine sahip olmadığını Yeniden doğallaştırmanın (yerine bozulması daha) çiftler protein ayrıştırılmasıyla bilinmeyen 33-35 kalır . Bu nedenle, biz ve diğerleri Hsp104 toksik preamyloid oligomerler ve amiloid fibriller 4,7,23,36-38 spesifik proteinlerin misfolding bağlı çeşitli nörodejeneratif hastalıklar için tedavi edici bir ajan olarak gelişmiş olabilir önerdi. Bu hastalıklar ile ilişkili toplanan türlerin doğrudan hedef tedaviler vardır. Ancak, Hsp104, Parkinson Hastalığı 23 gibi PrP 39 amiloid formları ile bağlı alfa-sinüklein oluşan toksik oligomerler ve amiloid fibriller çözünür . Daha da önemlisi, Hsp104 protein agregasyonunu azaltır ve Parkinson Hastalığı 23 ve Huntington hastalığı 38 kemirgen modellerinde nörodejenerasyon düzelir. İdeal olarak, tedavi optimize etmek ve yan etkileri en aza indirmek için, Hsp104 mühendislik ve seçici soru 4,7 hastalığın merkezi özel agrega bozulmasina potansiyelize olacaktır . Ancak, nasıl Hsp104 disaggregates sınırlı yapısal ve mekanik bir anlayış toplanan yapıları ve ilgisiz proteinler gibi farklı bir repertuar bu çalışmaların 30,40-42 köstekliyor . Hsp104 yapısı ve mekanizması anlamak için, saf protein ve sulandırmak, disaggregase aktivite minimal bileşenleri ile çalışmak için gerekli. Hsp104 ~ 5.3, ADP ve ATP varlığında veya yokluğunda 43-46 nükleotid yüksek protein konsantrasyonları hexamerizes pI 102kDa proteindir. Burada, son derece aktif, istikrarlı Hsp104 E. temizleme için optimize edilmiş bir protokol tarif coli. E. coli basitleştirilmiş büyük ölçekli üretim sağlar ve yöntemi çok sayıda Hsp104 varyantları için hızlı ve güvenilir bir şekilde yapılabilir. Bizim protokol Hsp104 saflığı artar ve E. önceki bir arıtma yöntemi ile karşılaştırıldığında, O&#39;nun 6-tag çıkarma işlemlerini basitleştiriyor coli, 47. Ayrıca, bizim protokol iki daha yeni protokoller 26,48 daha bayağı ve rahat.

Watch this Scientific Journal Video about Purification of Hsp104, a Protein Disaggregase at JoVE.com

Purification of Hsp104, a Protein Disaggregase

Burada, son derece aktif Hsp104 hexameric AAA + protein mayası, protein ayrıştırılmasıyla çiftler ATP hidrolizi arıtma için bir protokol açıklar. Bu düzeni yakınlık arıtma için His6 etiketli inşa patlatır<em&gt; E. coli</em&gt; Anyon-değiştirici kromatografi, TEV proteaz His6-tag kaldırılması, ve boyut dışlama kromatografisi izledi.

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 ...

purification-of-hsp104-a-protein-disaggregase

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17.3K Görüntüleme.  University of Pennsylvania. Burada, son derece aktif Hsp104 hexameric AAA + protein mayası, protein ayrıştırılmasıyla çiftler ATP hidrolizi arıtma için bir protokol açıklar. Bu düzeni yakınlık arıtma için His6 etiketli inşa patlatır E. coli Anyon-değiştirici kromatografi, TEV proteaz His6-tag kaldırılması, ve boyut dışlama kromatografisi izledi.

Video: Purification of Hsp104, a Protein Disaggregase

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

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

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

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

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum 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 ...

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 ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

Yüksek Aktif Maya Ribozomlar Kromatografik Arıtma

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

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 ...

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 ...

Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (A&beta;, tau), Parkinson's Disease (&alpha;-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms.
Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease1, 2, could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia3. Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C4, and that DISC1 aggresomes generated in vitro were cell-invasive5, similar to what had been shown for A&beta;6, tau7-9, &alpha;-synuclein10, polyglutamine11, or SOD1 aggregates12. These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders.
 Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic &alpha;-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.

The generation, purification and cell invasion of intracellular, cytoplasmic full length DISC1 protein aggresomes from cell cultures and of a labeled, multimeric recombinant DISC1 protein fragment in E. coli are described. Cell invasiveness is shown for recipient cells in cell culture and for neurons in vivo after stereotactical brain inoculation.

Hücre-invaziv DISC1 Protein Türlerinin Üretimi, Saflaştırılması, ve Karakterizasyonu

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases ...

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 ...

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. ...