Biz sinkrotron veri toplama sırasında destek için İsviçre Işık Kaynağı, Paul-Scherrer-Enstitüsü, Villigen, İsviçre&#39;de beamline X06DA personeli teşekkür ederiz. Biz mükemmel teknik destek için Monika Gompert minnettarız. Proje Köln Üniversitesi tarafından desteklenen ve Alman Araştırma Konseyi INST 216 / 682-1 FUGG hibe edildi. CP Kalkınma Sağlığı ve Hastalıkları Uluslararası Graduate School bir doktora bursu kabul edilmektedir. Bu sonuçlara yol açan bir araştırma hibe sözleşmesi No. 283570 (BioStruct-X) kapsamında Avrupa Topluluğu Yedinci Çerçeve Programı (FP7 / 2007-2013) fon aldı.

<ol>
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The authors have nothing to disclose.

X-ışını kristalografisi hala proteinlerin 28 üç boyutlu yakın atomik çözünürlükte yapılarını belirlemek için hızlı ve en doğru yöntemdir. Bununla birlikte, iyi sıralı tek kristallerinin büyümesini gerektirir. Bunlar genellikle almak zordur ve kristal devlet yapaydır. Bununla birlikte, diğer yöntemler ile belirlenmiş olan X-ışını kristalografisi ile tespit edilmiş protein yapılarının karşılaştırılması, özellikle NMR genellikle çok iyi bir uyum göstermektedir. PPEP-1 durumunda, bir NMR yapı S döngüsünün hareket dahil olmak üzere, kristal yapısı 14 en son 29 göstermektedir iyi bir uyum içinde yayınladı. Bu protokol, yapısal çalışmalar ve etiketsiz rPPEP-1 kristalleşmesi ve yapı tayini için N-terminal His-etiketli rPPEP-1 proteininin üretimini ve saf hale açıklanmaktadır. Tek kristaller Bu durumda büyümeye zor ve özel bir mikro-tohumlama işlemi gerektirmez. to bölümde aşağıdaki biz PPEP-1 sonuçlarını tartışmak ve protokol diğer herhangi bir proteinin üretimi ve kristalleşme için adapte olabilir nasıl gösterecektir. Yapı tasarımı ve ifade Üzerine Çeşitlemeler kristalleşme için nedeniyle kristalleştirme başarı ve protein yapısı 30 olan etkilerine sindirmek trombin ile His-tag çıkarılması tercih edilir olarak rPPEP-1, bir N-terminal His-etiketli varyantı (Şekil 1A) olarak ifade edilir. Diğer proteinler için, ya da bırakmak için N-terminal (ör vektörü pET22b ve 3 &#39;ucunda durma kodunu ile veya olmaksızın, bir ters primer aynı sınırlandırma bölgeleri kullanılarak klonlandı) ilave olarak bir Cı-terminal His-etiketli bir sürümünü test etmek uygun olabilir bir etiket kristalleşme sırasında yardımcı olabilecek bazı durumlarda olduğu gibi His-tag, bölünmemiş. C-terminal His-etiketli konstruktun rPPEP-1 verim ve kararlılık için daha düşüktü. En iyi s için Ayrıca, bir ilk testoluble protein ifadesi izlenerek E. coli suşlarının dahil edilmelidir: BL21 (DE3), BL21 (DE3) pLysS ya Lemo21 (DE3), BL21 (DE3) &#39;kodon artı RIPL ya Rosetta 2 (DE3), BL21 (DE3) Yıldız ve üç farklı sıcaklıkta / bekletme süreleri C41 (DE3) &#39;e (37 ° C, 5 ila 30 ° C&#39; de saat ve gece boyunca 20 ° C&#39;de 3-4 saat). Klonlanmadan önce, ilgi konusu proteinin bölünmesini engellemek için EXPASY PeptideCutter aracı 31 ile bir iç trombin bölünme sitesinin varlığı kontrol edin. Seçenek olarak ise, HRV3C için bir bölünme bölgesini sağlayan uygun vektörler bulunmaktadır (örneğin EMBL en pETM-14 32) ya da TEV proteaz (örn EMBL en pETM 11). Protein saflaştırma rPPEP-1 yapıları için hücre parçalama buz / su sonikasyon yoluyla yapılır. toplamak ya da bir hücre Topu kullanımı olabilir içeren daha &quot;yumuşak&quot; hücre parçalama yöntemi çöktürmek eğilimi daha duyarlı proteinler içind. Bu tür kimyasal hücre lizizi olarak liziz yöntemini kullanırken algılanmaz ilk santrifüj aşamasından sonra, çözünmeyen proteinlerin büyük miktarda kontrol edin. rPPEP-1 saflaştırılması için, genellikle Ninta reçine 2 ml kullanıldı. seçilen reçinenin protein bağlama kapasitesi ne kadar büyük üreticinin talimatları okuyun ve buna göre ayarlayın. RPPEP-1, reçinenin yalnızca 1 ml protein bir çok kolona değildi ve akış yoluyla (Şekil 1D) bulundu kullanıldı ilk arıtımında. Saflaştırılmış proteinin saflıkta alt olabilir çok reçine ile Öte yandan. Ninta matrise kullanılan His-işaretli konstruktun bağlanma afinitesi imidazol konsantrasyonu ayarlayın. Bir aşamalı yıkama prosedürü burada tercih edilir (örneğin, 10 mM, 30 mM, 50 mM, 70 mM imidazol) A280 izlenir ve her adım sırasında taban çizgisi ulaşmak için izin verilen. Bu şekilde, herhangi bir protein, bir esnasında elüt olur bile, kaybolur(50 ya da 70 mM gibi) yüksek imidazol yıkama adımı. durumunda USES-rPPEP-1 bazı protein zaten 30 mM imidazol de elutes, ama o temsil protein türünün ne tür belirsizdir. imidazol diyaliz veya protein çözeltiden seyreltilmekte Bazı protein / agrega hızlandırabilir. Bu durumda göre imidazol olmayan tampon 5-10 misli hacimde bir kap içine elüsyon ile imidazol konsantrasyonunu azaltmak, örneğin 150 mM elüsyon tamponu içinde imidazol konsantrasyonu ve yüksek imidazol konsantrasyonlarında maruz kalma süresini azaltmak planlanan elüsyon hacmi. gece boyunca 4 ° C&#39;de protein mg&#39;ı başına 2 adet trombin ile USES-rPPEP-1 inkübasyon için yaklaşık% 100 His-etiketi bölmek için yeterlidir. 20 ° C&#39;ye kadar 4 ° C&#39;de mg protein başına 1-10 birim kullanılarak ilgilenilen protein için trombinin miktarını ayarlayın. 5-10 dakika aralıklarla bir santrifüj ultrafiltrasyon ünitesi kullanılarak duraklama ve p karışımı proteinin konsantre zamanrotein deriştirme aygıtı içinde, kurma konsantrasyon gradyenti homojenleştirin. Aksi takdirde, yüksek ölçüde konsantre bir protein / çökelti bir araya olabilir. kristalleşme rPPEP-1 sürekli X-ışını kırınım analizi için uygun değildir yüksek koroziyona kristaller (tek kristaller, böylece birden fazla kristal kafeslerindeki sahip üst üste büyüyen) üretir. Açıklama çok çekirdeklenme olayları faz diyagramı (Şekil 7) çekirdeklenme bölgesinde gerçekleştiğini olabilir. <img alt="Şekil 7," src="/files/ftp_upload/55022/55022fig7.jpg" /> Şekil 7: bir protein kristalleştirme Deney faz diyagramı. Protein süper-doymuş olduğunda, kristaller sadece oluşturabilir. Çekirdeklenme metastabil zonun çekirdeklenme bölgesi ve kristal büyümesi meydana gelir. bir protein doygun olduğunda, açılan açık kalacaktır. &lt;olarak daha sonra rPPEP-1 herhangi bir çekirdeklenme durumunu iyileştirmek değil, protein veya Yağışlı konsantrasyonları düşürülmesi p class = &quot;jove_content&quot;&gt; artık görülmektedir ve damla açık kalmak. Küçük çekirdekler rPPEP-1 kristallerinin, sonunda büyüdüğü metastabil bölge (Şekil 7) doğrudan getirildiğinde mikro-tohumlama, tercih edilen yöntem olarak. Orijinal durumuna (2.4 M amonyum fosfat dibazik, 0.1 M Tris pH 8.5) konum C5 (Şekil 4) (pH ve yağışlı konsantrasyonu oldukça yüksek ucunda) yer aldığını bir şekilde yeni en optimizasyon ekranı tasarlayarak koşullar yarı kararlı bölge 16 (Şekil 7) bir kısmını temsil eden bir eğilimi yüksek olan düşük süperdoygunluk bir duruma karşılık gelir. Böylece, tohumlama sırasında getirilen çekirdekler potansiyel olarak büyük tek kristaller büyüyebilir. Orijinal tohum stoku 10 seyreltilmiş olabilir prosedürü (yeni oluşan kristallerin miktarı ve boyutu) optimize etmek <sup&gt; -2 10 -5. Burada tohum stoklarının birçok seyreltileri büyük tek kristallerinin üretimi için en iyi tohum seyreltme belirlemek için test edilebilir. Alternatif bir hayvan bıyık ya da kuyruk saç (tavşan, kedi, chinchilla ya da at) kullanarak çizgi tohumlama 33 istihdam edilebilir. Prosedür ürün peptidleri ve rPPEP-1 alt-tabaka-peptidler beraber-kristalize etmek için kullanılabilir. Optimizasyon prosedürü 250: 1.25 Å kadar kırıcı uzay grubu P2 1&#39;de Kristaller tohum stoklarının elde edilmiştir 1 seyreltilmiş. İki uzay grupları 1 2 1 2 1 (bağlanmamış protein) ve P2 1 (kompleks yapılar) P2 optimizasyon prosedürü amonyum fosfat ekran içinde çok benzer koşullar kaynaklanan ederken Kristaller, büyür. Bunların ikisi de R-PPEP-1 kristal formları, aktif sitenin prime yan ele esas olarak tüm küçük moleküller ve peptidleri bulunan kristal paket tek başına soake olabilirkristaller halinde d. molekülün bu yan kristal içinde bulunan çözücü madde erişilebilir. alt sitesi veya astarlanmış tarafının ele molekülleri S köprüsü açılması, rPPEP-1 ile birlikte kristalize olması gerekir ancak aktif sitesine bunlar için gerekli olacaktır. ayrıca 3 kalıntısı elde etmek, alt-tabaka, peptitlerin uzunluğu sınırlayan bir aslında - Buna ek olarak, rPPEP-1 S3&#39;site (bu alanda kristal kontakların teşviki) yakınında alt-tabaka bağlanma sitesi çıkışında komşu molekülleri tarafından temas ettirilir bu iki kristal formları prime yan. Kristal montaj ve cryo-koruma protein kristalleri montajı stereomicroscope altında manipülasyon bazı beceri gerektirir ve böylece biraz pratik gereken bir yöntemdir. Backgroun katkıda kristal etrafında çözücü fazlalığını önlemek için naylon döngü (veya dilediğiniz başka bir döngü, örneğin litholoop) optimal uzunluğunu seçinD saçılma ve kırınım verileri gürültü oranı, böylece daha düşük bir sinyal. Optimum döngü boyutu / uzunluğu da döngü arasından kayıp değil billur gibi kolay kristallerinin balıkçılık yapar. 100-200 um uzun boyutu olan rPPEP-1 kristaller için, 0.1-0.2 mm boyutlu naylon döngüler seçilmiştir. cryo-koruyucu da cryo-koşul transfer olduğunda kristal bir ozmotik şok karşılaşabilir olarak, veri kalitesinin kötüleşmesine katkıda bulunabilir. Bu engel, hatta kristal iç düzenini yok edebilir. Dikkatle cryo koruyucusunun türünü ve konsantrasyonunu seçin. rPPEP-1 kristalleri cryo-korunmuş 20% gliserol veya% 30 sukroz ile ya edildi. Başka bir ligand ile rPPEP-1 &#39;in bir alt-tabaka peptit kompleksi veya bir kompleks kristalize sükroz kriyo-koruyucu olarak seçilmelidir ise gliserol rPPEP-1 14 alt-tabaka bağlanma sitesinin prime siteye bağlanan olarak. yapı tayini Autosol çözümü 49.2 ± 18.4 bir BAYES-CC, 0.41 bir FOM (liyakat şekil), 0.17 bir çarpık ve 0.85 bir korelasyon RMS elde etti. Model haritası korelasyon 0.86 ve R / R serbest faktörler 0.21 / 0.24 bulunmaktadır. Tüm bu parametreler başarılı bir yapı çözeltisi gösterir.

Clostridium difficile, hastane antibiyotik ilişkili ishal enfeksiyonlarının 1 en önemli nedenlerinden biridir. Bu Gram-pozitif anaerobik bakteri dışkı-ağız yolu ile kendi spor formu yoluyla bulaşır. Son on yılda, yeni &#39;&#39; salgın &#39;&#39; ya da &#39;&#39; hipervirülan &#39;suşları (/ 027 örneğin BI / NAP1) Kuzey Amerika ve Avrupa&#39;da 2 yeni enfeksiyonların ve ölüm oranlarında ciddi bir artışa neden olmuştur. C. difficile -associated hastalığı (CDAD) yüksek ölüm oranları 3 ile hayatı tehdit eden kolon iltihabıdır. Belirtiler ishal 4&#39;ten psödomembranöz kolit 5 ve genellikle ölümcül zehirli megakolon 6 arasında değişir. Öldürücü suşlar Çok ilaca dirençli ve nüks oranı 7 yüksek olduğu gibi CDAD tedavisi zordur. anı tedavisi antibiyotikler metronidazol, fidaxomicin ya da vankomisin veya repetiti olarak içeren enf tekrarlayan olgular dışkı Mikrobiyota nakli. Yeni tedavi stratejileri acilen 8 ihtiyaç vardır. Bazı ilerlemeler C. difficile toksin B 9 hedefleyen terapötik monoklonal antikor Bezlotoxumab olarak kaydedilir, son zamanlarda başarılı bir faz III klinik çalışmalarda geçti ve FDA ve EMA ile onay için açılmıştı. Ek olarak, yeni antibiyotikler klinik çalışmalarda 10 farklı aşamalarında anda denenmektedir. yeni tedavi hedefleri belirlenmelidir etkili bir tedavi geliştirmek. (; CD2830 / Zmp1; PPEP-1 EC 3.4.24.89), C. difficile proteaz prolin-prolin endopeptidaz-1 keşfedilen bir knock-out suşunda PPEP-1 eksikliği gibi umut verici bir hedef olduğunu, C hastalık oluşturma azalır ., in vivo 11 difficile. PPEP-1 de C-terminalinde iki tane C. difficile adezinleri yaran bir salgılanmış metaloproteaz 12,13 olan 13 ve böylece yapışık Bacter serbestİnsan bağırsak epitelinden ia. Bu nedenle, C. difficile sesil ve hareketli fenotip arasındaki dengeyi koruyarak yer almaktadır. PPEP-1&#39;e karşı seçici inhibitörlerinin geliştirilmesi ve onun yüzeyler üç boyutlu yapının samimi bilgi vazgeçilmez olduğunu tanır anlamak. Bir alt-tabaka peptidi 14 PPEP-1 tek başına veya karmaşık ilk kristal yapışım çözdük. PPEP-1 ilk olarak bilinen proteaz, iki prolin kalıntıları 15 ila seçimli olarak böler peptid bağlar. Bu çift bükülmüş bir şekilde alt-tabakanın bağlanır ve proteaz aktif mevki 14 kapsar S döngüsünde yer alan tortuların uzun bir alifatik-aromatik ağ aracılığıyla stabilize eder. Bu alt-tabaka bağlanma modu PPEP-1&#39;e özgü olan ve bugüne kadar, insan proteazlar bulunamadı. Bu umut verici bir ilaç hedef haline getirir ve çok olası inhibitörlerinin hedef dışı etkileri. Ekran seçici PPEP-1 inh geliştirmek veibitors gelecek saf ve tek dağılımlı PPEP-1 proteininin büyük bir miktarı gereklidir. Bundan başka, tespit edilmesi gerekir PPEP-1 ilk önleyicileri, ko-kristal yapıları bağlanmasının modunu belirler. Bizim ellerde PPEP-1 sürekli iç içe gelişmiş kristaller üretir. Böylece PPEP-1 tek sapması kalitede kristaller üretmek için bir optimizasyon yöntemi geliştirmişlerdir. Bu protokolde, ayrıntılı PPEP-1 14 üretimi, saflaştırma, kristalleştirme ve Yapı çözümü tarif etmektedir. Bu salgılama sinyal dizisi, saflaştırma işareti kaldırılması ile afinite kromatografisi ve boyut çıkarma kromatografisine eksik PPEP-1 varyantın Escherichia coli hücre içi ifade kullanmak, çinko tek dalga boyu anormal dağılma vasıtasıyla bir optimizasyon ekran ve yapı belirlenmesi halinde 16 mikro-tohumlama ardından (çinko-SAD) 17. Bu protokol, (diğer proteinlerin üretimi ve yapı tayini için uyarlanabilir, örneğin &lt;/ Em&gt; metaloproteazlar) ile iç içe gelişmiş kristallerinin üretilmesi proteinler için, özellikle de. İstek üzerine, yapının DNA (pET28a-USES-rPPEP-1) ve difraksiyon verileri, eğitim amacıyla temin edilebilir.

<table><tbody><tr><td>&lt;strong&gt;Genes / Vectors / cell strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>pET28a vector</td><td>Merck-Millipore</td><td>69864</td><td>Thrombin cleavable N-terminal His-tag</td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; strain BL21 (DE3) Star</td><td>ThermoFisher Scientific</td><td>C601003</td><td>RNase H deficient</td></tr><tr><td>Codon-optimized gene (for &lt;em&gt;E. coli&lt;/em&gt;) of PPEP-1 (CD630_28300)</td><td>Geneart (Thermo Fisher Scientific)</td><td>custom</td><td>amino acids 27-220</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Yeast extract</td><td>any</td><td></td><td></td></tr><tr><td>Tryptone</td><td>any</td><td></td><td></td></tr><tr><td>Antifoam B</td><td>Sigma-Aldrich</td><td>A5757</td><td>aqueous-silicone emulsion</td></tr><tr><td>Agar</td><td>any</td><td></td><td></td></tr><tr><td>Kanamycin</td><td>any</td><td></td><td></td></tr><tr><td>IPTG</td><td>AppliChem</td><td>A1008</td><td></td></tr><tr><td>Tris-HCl</td><td>AppliChem</td><td>A1087</td><td>Buffer grade</td></tr><tr><td>NaCl</td><td>any</td><td></td><td>Buffer grade</td></tr><tr><td>DNaseI</td><td>AppliChem</td><td>A3778</td><td></td></tr><tr><td>Imidazole</td><td>AppliChem</td><td>A1073</td><td>Buffer grade</td></tr><tr><td>Thrombin</td><td>Sigma-Aldrich</td><td>T4648</td><td></td></tr><tr><td>Ammonium phosphate dibasic</td><td>Sigma-Aldrich</td><td>215996</td><td></td></tr><tr><td>Glycerol 100%</td><td>any</td><td></td><td>purest grade</td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>84097</td><td></td></tr><tr><td>Liquid nitrogen</td><td>any</td><td></td><td>for storage and cryocooling of crystals</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment (general)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Shaking incubator</td><td>any</td><td></td><td>providing temperatures of 20 &amp;deg;C - 37 &amp;deg;C</td></tr><tr><td>Glassware</td><td>any</td><td></td><td>baffled Erlenmeyer flasks (50 ml - 2.8 L)</td></tr><tr><td>Centrifuge for large culture volumes</td><td>any</td><td></td><td>centrifuge for processing volumes up to 12 L</td></tr><tr><td>Sonicator Vibra-Cell VCX500</td><td>Sonics</td><td>SO-VCX500</td><td>or any other sonicator / cell disruptor</td></tr><tr><td>Ultracentrifuge</td><td>any</td><td></td><td>centrifuge providing speeds up to 150,000 x g</td></tr><tr><td>NiNTA Superflow resin</td><td>Qiagen</td><td></td><td></td></tr><tr><td>Empty Glass Econo-Column</td><td>Bio-Rad</td><td>7371007</td><td>or any other empty glass or plastic column</td></tr><tr><td>Size exclusion chromatography column HiLoad Superdex 200 16/600</td><td>GE Healthcare</td><td>28989335</td><td></td></tr><tr><td>Chromatography system &amp;Auml;kta Purifier</td><td>GE Healthcare</td><td>28406264</td><td>or any other chromatography system</td></tr><tr><td>Dialysis tubing Spectra/Por 3</td><td>Spectrum Labs</td><td>132724</td><td></td></tr><tr><td>Dialysis tubing closures</td><td>Spectrum Labs</td><td>132738</td><td></td></tr><tr><td>Ultrafiltration units (concentrators) 10,000 NWCO</td><td>any</td><td></td><td></td></tr><tr><td>UV-Vis spectrophotometer</td><td>any</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment (crystallography)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Low volume pipette 0.1-10 &amp;micro;l</td><td>any</td><td></td><td></td></tr><tr><td>Positive displacement pipette Microman M10</td><td>Gilson</td><td>F148501</td><td></td></tr><tr><td>Crystallization robot</td><td>any</td><td></td><td></td></tr><tr><td>96-well crystallization plates TTP IQ with three protein wells</td><td>TTP</td><td>4150-05810</td><td>or any other 96-well crystallization plate&amp;nbsp;</td></tr><tr><td>24-well CombiClover Junior Plate</td><td>Jena Bioscience</td><td>EB-CJR</td><td></td></tr><tr><td>Crystal Clear Sealing Tape</td><td>Hampton Research</td><td>HR3-511</td><td></td></tr><tr><td>Siliconized Glass Cover Slides</td><td>Hampton Research</td><td>HR3-225</td><td></td></tr><tr><td>Commercial crystallization screens: SaltRx, Index, PEG/Ion, Crystal</td><td>Hampton Research</td><td>diverse</td><td></td></tr><tr><td>Commercial crystallization screens: Wizard, PACT++, JCSG++</td><td>Jena Bioscience</td><td>diverse</td><td></td></tr><tr><td>JBS Beads-for-Seeds</td><td>Jena Bioscience</td><td>CO-501</td><td></td></tr><tr><td>CrystalCap SPINE HT (nylon loops)</td><td>Hampton Research</td><td>diverse</td><td>loop sizes 0.025 mm - 0.5 mm</td></tr><tr><td>CrystalCap Vial</td><td>Hampton Research</td><td>HR4-904</td><td></td></tr><tr><td>Cryogenic Foam Dewar 800 ml</td><td>Hampton Research</td><td>HR4-673</td><td></td></tr><tr><td>Cryogenic Foam Dewar 2 L</td><td>Hampton Research</td><td>HR4-675</td><td></td></tr><tr><td>Vial Clamp, Straight</td><td>Hampton Research</td><td>HR4-670</td><td></td></tr><tr><td>CrystalWand Magnetic, Straight</td><td>Hampton Research</td><td>HR4-729</td><td></td></tr><tr><td>CryoCane 6 Vial Holder</td><td>Hampton Research</td><td>HR4-711</td><td></td></tr><tr><td>CryoSleeve</td><td>Hampton Research</td><td>HR4-708</td><td></td></tr><tr><td>CryoCane Color Coder - White</td><td>Hampton Research</td><td>HR4-713</td><td></td></tr><tr><td>Scalpel</td><td>any</td><td></td><td></td></tr><tr><td>Straight microforcep</td><td>any</td><td></td><td>for manipulation of sealing tape. &lt;em&gt;etc.&lt;/em&gt;</td></tr><tr><td>Acupuncture needle</td><td>any</td><td></td><td>&lt;em&gt;e.g.&lt;/em&gt; from a pharmacy</td></tr><tr><td>Stereo microscope</td><td>any</td><td></td><td>for inspection of crystallization plates and crystal mounting, magnification up to 160X</td></tr></tbody></table>

production, crystallization and structure determination of c. difficile ppep-1 via microseeding and zinc-sad

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a target for future development of inhibitors to decrease the virulence of the pathogen. To perform biophysical and structural characterization as well as inhibitor screening, large amounts of pure and active protein will be needed. We have developed a protocol for efficient production and purification of PPEP-1 by the use of E. coli as the expression host yielding sufficient amounts and purity of protein for crystallization and structure determination. Additionally, using microseeding, highly intergrown crystals of PPEP-1 can be grown to well-ordered crystals suitable for X-ray diffraction analysis. The methods could also be used to produce other recombinant proteins and to study the structures of other proteins producing intergrown crystals.

rPPEP-1, E. coli BL21 (DE3) Yıldız (Şekil 1C) en yüksek verimle çeşitli E.coli suşları aşırı eksprese edilir. İlk Ninta afinite kromatografi basamağından sonra, 6xHis-Tag başarılı protein çoğundan yanlabilir ve ikinci Ninta aşamada, düzenlenmemiş protein tam trombin sindirilmiş protein (Şekil 1D) ayrılabilir. Ara sıra muhtemelen dimerik türler (Şekil 2A) &#39;e uygun önü ile, monomer olarak bir S200 16/600 kolonu etiketlenmemiş rPPEP-1 taşınana üzerinde. Protein (Şekil 2B), saf ve verimi, E. coli kültürünün 1 L ila yaklaşık 50 mg bir proteindir. rPPEP-1 genellikle fosfat içeren, çeşitli koşullar (Şekil 3) kristalize olur. kristalleri yüksek tüm koşullarda iç içe gelişmiş, böylece kristal optimizasyonu yapılmalıdır zorunda edilir. Şekil 4, şematiktir tasviroptimizasyon prosedürünün e durumu ticari cam SaltRx E4 (52) (Şekil 3B-C) gelen kristaller için gerçekleştirilmiştir. Yine iç içe gelişmiş kristaller kuyular A6-D6 (Şekil 4) ortaya çıktı. Bir microseed hazır optimizasyonu ekranın durumu C6 (2.55 M amonyum fosfat dibazik, 8.5, 0.1 M Tris pH) yetiştirilen kristallerden hazırlanan tamponu 1: ana likör içinde 1.000. İki yedi gün açık yüksek kırınım kaliteli büyük tek kafes kristalleri çoğu damla (Şekil 5) gözlendi damla ile diğer kalan 20 koşullara seyreltilmiş tohum stoku ekim sonra. 1.67 Å çözünürlüğe naylon döngüler (Şekil 5E) kırınım verileri içine kristallerin monte edildikten sonra anormal bir sinyal 2 Å uzanan ile, çinko-SAD (Tablo 1) üzerinden yapı tayini için toplandı. Buna ek olarak, 1.4 çözünürlüğe ana difraksiyon verileri HIG belirlenmesi için toplandıH çözünürlüklü yapısı (Tablo 1). RPPEP-1 kristal yapısı (Şekil 6) ve R rafine doğal yapının modelini çözülebilir / 1.4 Å çözünürlükte 0.156 / 0.182 serbest faktörleri R (PDB ID: 5A0P) 14 (daha fazla arıtma için istatistik Tablosuna bakınız 1). <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Yapı / veri kümesi </td><td> zirve Zn-SAD </td><td> yerli </td></tr><tr><td> Veri koleksiyonu </td><td></td><td></td></tr><tr><td> Uzay grubu </td><td> P2 1 2 1 2 1 </td><td> P2 1 2 1 2 1 </td></tr><tr><td> A, B, C, (a) </td><td> 43.17, 71.68, 117,70 </td><td> 43.17, 71.77, 117,80 </td></tr><tr><td> α, β, γ (°) </td><td> 90, 90, 90 </td&gt; <td> 90, 90, 90 </td></tr><tr><td> Dalga boyu (A) </td><td> 1,28254 </td><td> 1 </td></tr><tr><td> Çözünürlük (Å) </td><td> 45.5-1.67 (1.72-1.67) </td><td> 45,5-1,40 (1,49-1,40) </td></tr><tr><td> gözlem sayısı </td><td> 779717 (34025) </td><td> 310074 (45851) </td></tr><tr><td> tek yansıma sayısı </td><td> 80960 (5597) </td><td> 71874 (11172) </td></tr><tr><td> çokluk </td><td> 9.6 (6.1) </td><td> 4.3 (4.1) </td></tr><tr><td> Bütünlük (%) </td><td> 99,2 (93,0) </td><td> 98.2 (95.6) </td></tr><tr><td> Rmerge (%) </td><td> 6.8 (56.5) </td><td> 5.2 (52.6) </td></tr><tr><td> &lt;I / σ (I) &#39;in&gt; </td><td> 21.86 (2.81) </td><td> 15.66 (2.48) </td></tr><tr><td> CC (1/2) (%) </td><td> 100 (85.1) </td><td> 99.9 (85.5) </td></tr><tr><td> Arıtma istatistikleri </td><td></td><td></td></tr><tr><td> Rwork / R ücretsiz d (%) </td><td></td><td> 15.60 / 18.17 </td></tr><tr><td> H olmayan protein atomlarının sayısı </td><td></td><td> 3109 </td></tr><tr><td> Su moleküllerinin sayısı </td><td></td><td> 532 </td></tr><tr><td> iyonları / ağır atomların sayısı </td><td></td><td> 2 Zn </td></tr><tr><td> diğer moleküllerin sayısı </td><td></td><td> - </td></tr><tr><td> TLS gruplarının sayısı / zincir </td><td></td><td> 9 </td></tr><tr><td> Kök ortalama kare sapma </td><td></td><td></td></tr><tr><td> Bağ uzunlukları (Å) </td><td></td><td> 0.006 </td></tr><tr><td> Bağ açıları (°) </td><td></td><td> 1.022 </td></tr><tr><td> Ortalama B faktörü (Å 2) </td><td></td><td></td></tr><tr><td> Tüm protein atomuna </td><td></td><td> 16.12 </td></tr><tr><td> Su </td><td></td><td> 29.74 </td></tr><tr><td> diğer atomlar </td><td></td><td> 10.12 </td></tr><tr><td> Ramachandran arsa e (%) </td><td></td><td></td></tr><tr><td> En çok tercih edilen </td><td></td><td> 98,72 </td></tr><tr><td> Ayrıca izin </td><td></td><td> 1.28 </td></tr><tr><td> İzin verilmeyen </td><td></td><td> 0 </td></tr><tr><td> PDB giriş </td><td></td><td> 5a0p </td></tr></tbody></table> Tablo 1: Veri toplama ve arıtma istatistikleri.

Watch this Scientific Journal Video about Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD at JoVE.com

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Proline-proline endopeptidase-1 (PPEP-1) is a secreted metalloprotease and promising drug-target from the human pathogen Clostridium difficile. Here we describe all methods necessary for the production and structure determination of this protein.

Üretim, Kristalleşme ve Yapı Tayini ve<em&gt; C. difficile</em&gt; PPEP-1 mikro-tohumlama ve Çinko-SAD aracılığıyla

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a ...

production-crystallization-structure-determination-c-difficile-ppep-1

Nanyang Technological University

Research

JoVE Journal

Biochemistry

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

11.5K Görüntüleme.  University of Cologne. Proline-proline endopeptidase-1 (PPEP-1) is a secreted metalloprotease and promising drug-target from the human pathogen Clostridium difficile. Here we describe all methods necessary for the production and structure determination of this protein. 

Video: Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.

Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

Yapısal Çalışmaları üretimi disülfid stabilize Transmembran Peptit Kompleksi

Physical  interactions among the lipid-embedded alpha-helical domains of membrane  proteins play a crucial role in folding and assembly of membrane ...

Kimya

Solid-phase Submonomer Synthesis of Peptoid Polymers and their Self-Assembly into Highly-Ordered Nanosheets

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and fold into higher order nanostructures. Structurally similar to peptides, peptoids are poly N-substituted glycines, where the side chains are attached to the nitrogen rather than the alpha-carbon. Their ease of synthesis and structural diversity allows testing of basic design principles to drive de novo design and engineering of new biologically-active and nanostructured materials.
Here, a simple manual peptoid synthesis protocol is presented that allows the synthesis of long chain polypeptoids ( up to 50mers) in excellent yields. Only basic equipment, simple techniques (e.g. liquid transfer, filtration), and commercially available reagents are required, making peptoids an accessible addition to many researchers' toolkits. The peptoid backbone is grown one monomer at a time via the submonomer method which consists of a two-step monomer addition cycle: acylation and displacement. First, bromoacetic acid activated in situ with N,N'-diisopropylcarbodiimide acylates a resin-bound secondary amine. Second, nucleophilic displacement of the bromide by a primary amine follows to introduce the side chain. The two-step cycle is iterated until the desired chain length is reached. The coupling efficiency of this two-step cycle routinely exceeds 98% and enables the synthesis of peptoids as long as 50 residues. Highly tunable, precise and chemically diverse sequences are achievable with the submonomer method as hundreds of readily available primary amines can be directly incorporated.
 
Peptoids are emerging as a versatile biomimetic material for nanobioscience research because of their synthetic flexibility, robustness, and ordering at the atomic level. The folding of a single-chain, amphiphilic, information-rich polypeptoid into a highly-ordered nanosheet was recently demonstrated. This peptoid is a 36-mer that consists of only three different commercially available monomers: hydrophobic, cationic and anionic. The hydrophobic phenylethyl side chains are buried in the nanosheet core whereas the ionic amine and carboxyl side chains align on the hydrophilic faces. The peptoid nanosheets serve as a potential platform for membrane mimetics, protein mimetics, device fabrication, and sensors. Methods for peptoid synthesis, sheet formation, and microscopy imaging are described and provide a simple method to enable future peptoid nanosheet designs.

A simple and general manual peptoid synthesis method involving basic equipment and commercially available reagents is outlined, enabling peptoids to be easily synthesized in most laboratories. The synthesis, purification and characterization of an amphiphilic peptoid 36mer is described, as well as its self-assembly into highly-ordered nanosheets.

Yüksek Sıralı Nanosheets Peptoid Polimerler ve Self-Meclis Solid-faz Submonomer Sentezi

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and ...

Biyomühendislik

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant strains. Carbohydrate substrate binding proteins (SBPs) represent viable targets for the development of protein-based vaccines and new antimicrobials because of their extracellular localization and the centrality of carbohydrate import for pneumococcal metabolism, respectively. Described here is a rationalized integrated protocol to carry out a comprehensive characterization of SP0092, which can be extended to other carbohydrate SBPs from the pneumococcus and other bacteria. This procedure can aid the structure-based design of inhibitors for this class of proteins. Presented in the first part of this manuscript are protocols for biochemical analysis by thermal shift assay, multi angle light scattering (MALS), and size exclusion chromatography (SEC), which optimize the stability and homogeneity of the sample directed to crystallization trials and so enhance the probability of success. The second part of this procedure describes the characterization of the SBP crystals using a tunable wavelength anomalous diffraction synchrotron beamline, and data collection protocols for measuring data that can be used to resolve the crystallized protein structure.

A streamlined protocol for performing an extensive biochemical and structural characterization of a carbohydrate substrate binding protein from Streptococcus pneumoniae is presented.

Karbonhidrat taşıma substrat-bağlayıcı-protein SP0092 biyokimyasal ve yapısal karakterizasyonu

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

Biyokimya

Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States alone, an average of 41,400 deaths and 1.86 million hospitalizations are caused by influenza virus infection each year 1. Point mutations in the polymerase basic protein 2 subunit (PB2) have been linked to the adaptation of the viral infection in humans 2. Findings from such studies have revealed the biological significance of PB2 as a virulence factor, thus highlighting its potential as an antiviral drug target.
The structural genomics program put forth by the National Institute of Allergy and Infectious Disease (NIAID) provides funding to Emerald Bio and three other Pacific Northwest institutions that together make up the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The SSGCID is dedicated to providing the scientific community with three-dimensional protein structures of NIAID category A-C pathogens. Making such structural information available to the scientific community serves to accelerate structure-based drug design.
Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for new lead discovery by targeting a pathway or an entire protein family. Emerald Bio has developed a high-throughput, multi-target parallel processing pipeline (MTPP) for gene-to-structure determination to support the consortium. Here we describe the protocols used to determine the structure of the PB2 subunit from four different influenza A strains.

Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for lead discovery. The following article highlights how the Seattle Structural Genomics Center for Infectious Disease utilizes a multi-target approach for gene-to-structure determination of the PB2 influenza A subunit.

Grip Polimeraz Gen-to-yapı belirlenmesi PB2 alt birimi için çoklu hedef Paralel İşleme Yaklaşımı

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States ...

İmmünoloji ve Enfeksiyon

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation.

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a pathogenic bacterium that is a strict anaerobe and causes antibiotic associated diarrhea (AAD). Here, methods for isolating, culturing and maintaining C. difficile vegetative cells and spores are described. These techniques necessitate an anaerobic chamber, which requires regular maintenance to ensure proper conditions for optimal C. difficile cultivation.

Kültürleme ve bakımı<em&gt; Clostridium difficile</em&gt; Bir Anaerobik Çevre

Clostridium  difficile is a Gram-positive, anaerobic, sporogenic bacterium  that is primarily responsible for antibiotic associated diarrhea (AAD) and is ...

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion cofactors triggering a dimer-dodecamer transition. Upon metal ion binding, several structural modifications occur in the active site and at the interaction interface, shaping dimers to promote the self-assembly. To observe such modifications, stable oligomers must be isolated prior to structural study. Reported here is a method that allows the purification of stable dodecamers and dimers of TmPep1050, an M42 aminopeptidase of T. maritima, and their structure determination by X-ray crystallography. Dimers were prepared from dodecamers by removing metal ions with a chelating agent. Without their&#160;cofactor, dodecamers became less stable and were fully dissociated upon heating. The oligomeric structures were solved by the straightforward molecular replacement approach. To illustrate the methodology, the structure of a TmPep1050 variant, totally impaired in metal ion binding, is presented showing no further breakdown of dimers to monomers.

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

This protocol has been developed to study the dimer-dodecamer transition of TmPep1050, an M42 aminopeptidase, at the structural level. It is a straightforward pipeline starting from protein purification to X-ray data processing. Crystallogenesis, data set indexation, and molecular replacement have been emphasized through a case of study, TmPep1050H60A H307A variant.

X-Ray Kristalografi Thermotoga maritima M42 Aminopeptidaz TmPep1050 Oligomerik Devlet Geçiş Çalışma

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion ...

Optimizing the Growth of Endothiapepsin Crystals for Serial Crystallography Experiments

Here, a protocol is presented to facilitate the creation of large volumes (&#62; 100 &#181;L) of micro-crystalline slurries suitable for serial crystallography experiments at both synchrotrons and XFELs. The method is based upon an understanding of the protein crystal phase diagram, and how that knowledge can be utilized. The method is divided into three stages: (1) optimizing crystal morphology, (2) transitioning to batch, and (3) scaling. Stage 1 involves finding well diffracting, single crystals, hopefully but not necessarily, presenting in a cube-like morphology. In Stage 2, the Stage 1 condition is optimized by crystal growth time. This strategy can transform crystals grown by vapor diffusion to batch. Once crystal growth can occur within approximately 24 h, a morphogram of the protein and precipitant mixture can be plotted and used as the basis for a scaling strategy (Stage 3). When crystals can be grown in batch, scaling can be attempted, and the crystal size and concentration optimized as the volume is increased. Endothiapepsin has been used as a demonstration protein for this protocol. Some of the decisions presented are specific to endothiapepsin. However, it is hoped that the way they have been applied will inspire a way of thinking about this procedure that others can adapt to their own projects.

The aim of this article is to give the viewer a solid understanding of how to transform their small-volume, vapor-diffusion protocol, for growing large, single protein crystals, into a large-volume batch micro-crystallization method for serial crystallography.

Seri Kristalografi Deneyleri için Endotiapepsin Kristallerinin Büyümesini Optimize Etme

Here, a protocol is presented to facilitate the creation of large volumes (&#62; 100 &#181;L) of micro-crystalline slurries suitable for serial ...

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We describe a versatile microfluidic chip that enables the production of crystals by the efficient method of counter-diffusion. The convection-free environment provided by the microfluidic channels is ideal for crystal growth and useful to diffuse a substrate into the active site of the crystalline enzyme. Here we applied this approach to the CCA-adding enzyme of the psychrophilic bacterium Planococcus halocryophilus in the presented example. After crystallization and substrate diffusion/soaking, the crystal structure of the enzyme:substrate complex was determined at room temperature by serial crystallography and the analysis of multiple crystals directly inside the chip. The whole procedure preserves the genuine diffraction properties of the samples because it requires no crystal handling.

A versatile microfluidic device is described that enables the crystallization of an enzyme using the counter-diffusion method, the introduction of a substrate in the crystals by soaking, and the 3D structure determination of the enzyme:substrate complex by a serial analysis of crystals inside the chip at room temperature.

Çok Yönlü Bir Mikroakışkan Çipte Seri Kristalografi ile Bir Enzim:Substrat Kompleksinin Kristalizasyonu ve Yapısal Belirlenmesi

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We ...

Detecting Cortex Fragments During Bacterial Spore Germination

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming bacterium, Bacillus subtilis. Germination is triggered by small molecule germinants and occurs without the need for macromolecular synthesis. Though differences exist between the mechanisms of spore germination in species of Bacillus and Clostridium, a common requirement is the hydrolysis of the peptidoglycan-like cortex which allows the spore core to swell and rehydrate. After rehydration, metabolism can begin and this, eventually, leads to outgrowth of a vegetative cell. The detection of hydrolyzed cortex fragments during spore germination can be difficult and the modifications to the previously described assays can be confusing or difficult to reproduce. Thus, based on our recent report using this assay, we detail a step-by-step protocol for the colorimetric detection of cortex fragments during bacterial spore germination.

Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.

Bakteriyel Spor Filizlenmesinin sırasında Cortex Fragments algılama

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming ...

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of &#947;-32-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for characterizing the activity of any enzyme that hydrolyzes a nucleotide triphosphate bond or transfers a terminal phosphate from a phosphate donor to another molecule.

A Purification and In Vitro Activity Assay for a pppGpp Synthetase from Clostridium difficile

Here, we describe a method for purifying histidine-tagged pyrophosphokinase enzymes and utilizing thin layer chromatography of radiolabelled substrates and products to assay for the enzymatic activity in vitro. The enzyme activity assay is broadly applicable to any kinase, nucleotide cyclase, or phosphor-transfer reaction whose mechanism includes nucleotide triphosphate hydrolysis.

Bir arınma ve In Vitro etkinliği tahlil için (p) ppGpp sentetaz Clostridium difficile üzerinden

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to ...

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