Name: production, crystallization and structure determination of c. difficile ppep-1 via microseeding and zinc-sad
Uploaded: 2016-12-30T15:00:00.000+00:00
Duration: 13 min 34 s
Description: Watch this Scientific Journal Video about Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD at JoVE.com

Ringraziamo il personale del X06DA linea di luce alla sorgente Swiss Light, Paul-Scherrer-Institute, Villigen, Svizzera per il supporto durante la raccolta dei dati di sincrotrone. Siamo grati a Monika Gompert per un eccellente supporto tecnico. Il progetto è stato sostenuto dall&#39;Università di Colonia e concedere INST 216 / 682-1 FUGG dal Consiglio di ricerca tedesco. Una borsa di studio di dottorato presso la Graduate School Internazionale in Sviluppo salute e malattia a CP è riconosciuto. La ricerca che ha portato a questi risultati è stata finanziata dal Settimo Programma Quadro della Comunità Europea (FP7 / 2007-2013), contratto di sovvenzione n ° 283.570 (BioStruct-X).

<ol>
	<li>Bouza, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+Clostridium+difficile+infection:+understanding+the+healthcare+burden.">Consequences of Clostridium difficile infection: understanding the healthcare burden.</a> Clin Microbiol Infect. 18 (Suppl 6), 5-12 (2012).</li><li>O'Connor, J. R., Johnson, S., Gerding, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clostridium+difficile+infection+caused+by+the+epidemic+BI/NAP1/027+strain.">Clostridium difficile infection caused by the epidemic BI/NAP1/027 strain.</a> Gastroenterology. 136 (6), 1913-1924 (2009).</li><li>Mitchell, B. G., Gardner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mortality+and+Clostridium+difficile+infection:+a+review.">Mortality and Clostridium difficile infection: a review.</a> Antimicrob Resist Infect Control. 1 (1), (2012).</li><li>George, W. L., Sutter, V. L., Finegold, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+agent-induced+diarrhea--a+bacterial+disease.">Antimicrobial agent-induced diarrhea--a bacterial disease.</a> J Infect Dis. 136 (6), 822-828 (1977).</li><li>George, R. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Clostridium+difficile+as+a+cause+of+pseudomembranous+colitis.">Identification of Clostridium difficile as a cause of pseudomembranous colitis.</a> Br Med J. 1 (6114), 695 (1978).</li><li>Bartlett, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Narrative+review:+the+new+epidemic+of+Clostridium+difficile-associated+enteric+disease.">Narrative review: the new epidemic of Clostridium difficile-associated enteric disease.</a> Ann Intern Med. 145 (10), 758-764 (2006).</li><li>Kelly, C. P., LaMont, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clostridium+difficile--more+difficult+than+ever.">Clostridium difficile--more difficult than ever.</a> N Engl J Med. 359 (18), 1932-1940 (2008).</li><li>&#220;nal, C. M., Steinert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+therapeutic+strategies+for+Clostridium+difficile+infections.">Novel therapeutic strategies for Clostridium difficile infections.</a> Expert Opin Ther Targets. 20 (3), 269-285 (2016).</li><li>Kelly, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Monoclonal+Antibody,+Bezlotoxumab+Targeting+C.+difficile+Toxin+B+Shows+Efficacy+in+Preventing+Recurrent+C.+difficile+Infection+(CDI)+in+Patients+at+High+Risk+of+Recurrence+or+of+CDI-Related+Adverse+Outcomes.">The Monoclonal Antibody, Bezlotoxumab Targeting C. difficile Toxin B Shows Efficacy in Preventing Recurrent C. difficile Infection (CDI) in Patients at High Risk of Recurrence or of CDI-Related Adverse Outcomes.</a> Gastroenterology. 150 (4), S122 (2016).</li><li>Tsutsumi, L. S., Owusu, Y. B., Hurdle, J. G., Sun, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+the+discovery+of+treatments+for+C.+difficile+infection:+A+clinical+and+medicinal+chemistry+review.">Progress in the discovery of treatments for C. difficile infection: A clinical and medicinal chemistry review.</a> Curr Top Med Chem. 14 (1), 152-175 (2014).</li><li>Hensbergen, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clostridium+difficile+secreted+Pro-Pro+endopeptidase+PPEP-1+(ZMP1/CD2830)+modulates+adhesion+through+cleavage+of+the+collagen+binding+protein+CD2831.">Clostridium difficile secreted Pro-Pro endopeptidase PPEP-1 (ZMP1/CD2830) modulates adhesion through cleavage of the collagen binding protein CD2831.</a> FEBS Lett. 589 (24), 3952-3958 (2015).</li><li>Cafardi, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+zinc+metalloprotease+through+a+global+analysis+of+Clostridium+difficile+extracellular+proteins.">Identification of a novel zinc metalloprotease through a global analysis of Clostridium difficile extracellular proteins.</a> PLoS One. 8 (11), e81306 (2013).</li><li>Hensbergen, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+secreted+metalloprotease+(CD2830)+from+Clostridium+difficile+cleaves+specific+proline+sequences+in+LPXTG+cell+surface+proteins.">A novel secreted metalloprotease (CD2830) from Clostridium difficile cleaves specific proline sequences in LPXTG cell surface proteins.</a> Mol Cell Proteomics. 13 (5), 1231-1244 (2014).</li><li>Schacherl, M., Pichlo, C., Neundorf, I., Baumann, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Basis+of+Proline-Proline+Peptide+Bond+Specificity+of+the+Metalloprotease+Zmp1+Implicated+in+Motility+of+Clostridium+difficile.">Structural Basis of Proline-Proline Peptide Bond Specificity of the Metalloprotease Zmp1 Implicated in Motility of Clostridium difficile.</a> Structure. 23 (9), 1632-1642 (2015).</li><li>Rawlings, N. D., Waller, M., Barrett, A. J., Bateman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MEROPS:+the+database+of+proteolytic+enzymes,+their+substrates+and+inhibitors.">MEROPS: the database of proteolytic enzymes, their substrates and inhibitors.</a> Nucleic Acids Res. 42 (Release 10.0), D503-D509 (2014).</li><li>Bergfors, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeds+to+crystals.">Seeds to crystals.</a> J Struct Biol. 142 (1), 66-76 (2003).</li><li>Dauter, Z., Dauter, M., Dodson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jolly+SAD.">Jolly SAD.</a> Acta Crystallogr D Biol Crystallogr. 58 (Pt 3), 494-506 (2002).</li><li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227 (5259), 680-685 (1970).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal Biochem. 72, 248-254 (1976).</li><li>Kabsch, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XDS.">XDS.</a> Acta Crystallogr D Biol Crystallogr. 66 (Pt 2), 125-132 (2010).</li><li>Adams, P. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PHENIX:+a+comprehensive+Python-based+system+for+macromolecular+structure+solution.">PHENIX: a comprehensive Python-based system for macromolecular structure solution.</a> Acta Crystallogr D Biol Crystallogr. 66, 213-221 (2010).</li><li>Emsley, P., Lohkamp, B., Scott, W. G., Cowtan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+and+development+of+Coot.">Features and development of Coot.</a> Acta Crystallogr D Biol Crystallogr. 66 (Pt 4), 486-501 (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The PyMOL Molecular Graphics System. , (2002).</li><li>Pettersen, E. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UCSF+Chimera--a+visualization+system+for+exploratory+research+and+analysis.">UCSF Chimera--a visualization system for exploratory research and analysis.</a> J Comput Chem. 25 (13), 1605-1612 (2004).</li><li>Wang, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolution+of+phase+ambiguity+in+macromolecular+crystallography.">Resolution of phase ambiguity in macromolecular crystallography.</a> Methods Enzymol. 115, 90-112 (1985).</li><li>McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C., Read, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phaser+crystallographic+software.">Phaser crystallographic software.</a> J Appl Crystallogr. 40 (Pt 4), 658-674 (2007).</li><li>Zwart, P. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+structure+solution+with+the+PHENIX+suite.">Automated structure solution with the PHENIX suite.</a> Methods Mol Biol. 426, 419-435 (2008).</li><li>Zheng, H., Handing, K. B., Zimmerman, M. D., Shabalin, I. G., Almo, S. C., Minor, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-ray+crystallography+over+the+past+decade+for+novel+drug+discovery+-+where+are+we+heading+next?.">X-ray crystallography over the past decade for novel drug discovery - where are we heading next?.</a> Expert Opin Drug Discov. 10 (9), 975-989 (2015).</li><li>Rubino, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+characterization+of+zinc-bound+Zmp1,+a+zinc-dependent+metalloprotease+secreted+by+Clostridium+difficile.">Structural characterization of zinc-bound Zmp1, a zinc-dependent metalloprotease secreted by Clostridium difficile.</a> J Biol Inorg Chem. 21 (2), 185-196 (2016).</li><li>Carson, M., Johnson, D. H., McDonald, H., Brouillette, C., Delucas, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=His-tag+impact+on+structure.">His-tag impact on structure.</a> Acta Crystallogr D Biol Crystallogr. 63 (Pt 3), 295-301 (2007).</li><li>Gasteiger, E., Walker, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Identification+and+Analysis+Tools+on+the+ExPASy+Server.">Protein Identification and Analysis Tools on the ExPASy Server.</a> The Proteomics Protocols Handbook. , 571-607 (2005).</li><li>Dummler, A., Lawrence, A. M., de Marco, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+screening+for+the+detection+of+soluble+fusion+constructs+expressed+in+E.+coli+using+a+modular+set+of+vectors.">Simplified screening for the detection of soluble fusion constructs expressed in E. coli using a modular set of vectors.</a> Microb Cell Fact. 4, 34 (2005).</li><li>Stura, E. A., Wilson, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+the+streak+seeding+technique+in+protein+crystallization.">Applications of the streak seeding technique in protein crystallization.</a> J Crys Growth. 110 (1), 270-282 (1991).</li></ol>

The authors have nothing to disclose.

Cristallografia a raggi X è ancora il metodo più veloce e più accurato per determinare tridimensionali strutture risoluzione quasi atomica delle proteine 28. Tuttavia, esso richiede la crescita di cristalli singoli ben ordinate. Questi sono spesso difficili da ottenere e stato cristallino è artificiale. Tuttavia, un confronto di strutture proteiche determinata mediante cristallografia a raggi X con quelle determinate con altri metodi, in particolare NMR, generalmente mostra un ottimo accordo. Nel caso di ...

Clostridium difficile è una delle principali cause di infezioni nosocomiali diarrea associata agli antibiotici 1. Questo batterio anaerobico Gram-positivi si trasmette attraverso la sua forma di spore per via fecale-orale. Negli ultimi dieci anni, il nuovo &#39;&#39; epidemia &#39;&#39; o &#39;&#39; ipervirulento &#39;&#39; ceppi (ad es BI / NAP1 / 027) ha causato un drastico aumento delle nuove infezioni e il tasso di mortalità in Nord America e in Europa 2. C. malattia -associated difficile (CDAD) è un pericolo di vita infiammazione del colon con alti tassi di mortalità 3. I sintomi variano da diarrea 4 a colite pseudomembranosa 5 e il megacolon tossico spesso fatale 6. Il trattamento di CDAD è difficile in quanto i ceppi virulenti sono multi-resistente e il tasso di recidiva è alto 7. Al momento terapia include il metronidazolo antibiotici, fidaxomicina o vancomicina, o in repetitiVely casi ricorrenti di trapianto microbiota fecale. Nuove strategie terapeutiche sono urgentemente necessari 8. Alcuni progressi viene registrato come il anticorpo monoclonale terapeutico Bezlotoxumab, il targeting tossina di C. difficile B 9, ha recentemente superato con successo di fase III di sperimentazione clinica ed è stato depositato per l&#39;approvazione con la FDA ed EMA. Inoltre, nuovi antibiotici sono in fase di sperimentazione in questo momento nelle diverse fasi della sperimentazione clinica 10. Per sviluppare un trattamento efficace devono essere identificati nuovi bersagli terapeutici. La recente scoperta da C. difficile proteasi endopeptidasi-1 prolina-prolina (PPEP-1; CD2830 / Zmp1; EC 3.4.24.89) è un obiettivo così promettente, come la mancanza di PPEP-1 in un ceppo knock-out diminuisce la virulenza di C . difficile in vivo 11. PPEP-1 è una metalloproteasi secreta 12,13 fendendo due adesine C. difficile al loro C-terminale 13 liberando così il Bacter aderentiia dal epitelio intestinale umano. Pertanto, è coinvolto nel mantenere l&#39;equilibrio tra il fenotipo sessili e motilità di C. difficile. Per sviluppare inibitori selettivi contro PPEP-1 e per capire come riconosce i suoi substrati intima conoscenza della sua struttura tridimensionale è indispensabile. Abbiamo risolto la prima struttura cristallina di PPEP-1 solo e in complesso con un peptide substrato 14. PPEP-1 è la proteasi prima noto che selettivamente scinde i legami peptidici tra due residui di prolina 15. Si lega il substrato in doppio-piegato e stabilizza tramite una rete alifatico-aromatici estesa di residui ubicata nella S-ciclo che copre la proteasi sito attivo 14. Questa modalità substrato vincolante riservate PPEP-1 e non trovato in proteasi umane fino ad oggi. Questo lo rende un bersaglio promettente farmaco, e off-bersaglio effetti degli inibitori molto improbabili. Per sviluppare e schermo selettivo PPEP-1 inhibitors in futuro è necessario una grande quantità di puro e monodisperse PPEP-1 proteina. Inoltre, per determinare la modalità di legame dei primi inibitori, strutture co-cristalline con PPEP-1 dovrà essere determinato. Nelle nostre mani PPEP-1 produce costantemente cristalli intergrown. Così abbiamo sviluppato una procedura di ottimizzazione per produrre cristalli singoli di diffrazione qualità di PPEP-1. In questo protocollo si descrive in dettaglio la soluzione di produzione, purificazione, cristallizzazione e la struttura di PPEP-1 14. Usiamo espressione intracellulare in Escherichia coli di una variante PPEP-1 privo della sequenza segnale di secrezione, cromatografia di affinità e cromatografia ad esclusione sterica con la rimozione del tag purificazione, seguita da microseeding 16 in uno schermo ottimizzazione e determinazione della struttura tramite zinco singola lunghezza d&#39;onda di dispersione anomala (zinco-SAD) 17. Questo protocollo può essere adattato per la determinazione della struttura produttiva e di altre proteine (ad esempio &lt;/ Em&gt; metalloproteasi), in particolare per le proteine ​​che producono cristalli intergrown. Su richiesta, il plasmide DNA del costrutto (pET28a-NHIS-rPPEP-1) dati di diffrazione e può essere fornita per scopi didattici.

<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 è sovraespresso in molti ceppi di E. coli, con il più alto rendimento in E. coli BL21 (DE3) stella (Figura 1C). Dopo la prima fase di cromatografia di affinità NiNTA la 6xHis-tag può essere scisso con successo fuori dalla maggior parte delle proteine e nella seconda fase NiNTA proteina non digerita può essere completamente separato dal trombina digerito proteine (Figura 1D). Su un S200 16/600 colonna senza tag rPPEP-1 migra...

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.

Produzione, Cristallizzazione e struttura Determinazione<em&gt; C. difficile</em&gt; PPEP-1 tramite Microseeding e Zinco-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 ...

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

Research

JoVE Journal

Biochemistry

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

11.5K Views.  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.

Produzione di disolfuro-stabilizzati peptide transmembrana per gli studi strutturali

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

Ricerca

Chimica

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.

Sintesi in fase solida Submonomer dei Polimeri Peptoid e il loro self-assembly in altamente ordinato nanosheets

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

Bioingegneria

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.

Caratterizzazione biochimica e strutturale del carboidrato trasporto substrato-proteina-SP0092

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

Biochimica

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.

Multi-target Parallel Processing Approccio per Gene-to-struttura Determinazione della polimerasi Influenza PB2 subunità

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

Immunologia e infezioni

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.

Coltura e manutenzione<em&gt; Clostridium difficile</em&gt; In un ambiente anaerobico

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.

Cristallografia a raggi X per studiare la transizione di stato oligomerica della termotoga maritima M42 Aminopeptidasi TmPep1050

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.

Ottimizzazione della crescita dei cristalli di endotiapepsina per esperimenti di cristallografia seriale

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.

Cristallizzazione e determinazione strutturale di un enzima:complesso di substrati mediante cristallografia seriale in un chip microfluidico versatile

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.

Rilevamento Cortex Frammenti Durante la germinazione delle spore batteriche

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.

Una purificazione e l'analisi di attività In Vitro per un ppGpp (p) sintetasi da Clostridium difficile

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

Produzione, Cristallizzazione e struttura Determinazione

Riepilogo

Esplora Altri Video

Capitoli in questo video