The funding for this study was provided by the CHS foundation. We acknowledge the protein crystallization platform within the excellence cluster CellNetworks of the University of Heidelberg for crystal screening and the European Synchrotron Radiation Facility for provision of synchrotron radiation facilities.

1. P dominio Clonazione <ol><li> Determinare la regione codificante dominio P per l&#39;allineamento di sequenze di ceppi di norovirus (ad esempio, ceppo GII.10, GenBank: AF504671, PDB-ID: 3ONU) 6. Inoltre, rimuovere la regione flessibile all&#39;estremità C-terminale del dominio P (Figura 2A). Codone-ottimizzare il DNA per E. espressione coli e comprendono BamHI (N-terminale) e NotI (C-terminale) siti di restrizione al fine di sub-clone del dominio P regione codificante in pMalc2x vettore di espressione 6,7. Nota: La regione codificante dominio P è ottimizzata e sintetizzato da un servizio commerciale. Il dominio P regione codificante (inserto) è di circa 1 kb di lunghezza e consegnato in un vettore di trasferimento standard. </li><li> Digest 2 ug del vettore di trasferimento con ogni 1 BamHI microlitri (20.000 U / ml) e NotI (10.000 U / ml) enzimi di restrizione per 1 ora a 37 ° C con il produttore fornito buffer. </li><li> Separare l&#39;inserto digerito su un gel di agarosio 1%per 20 min a 135 V e purificare il DNA dell&#39;inserto dal gel usando un kit commerciale. </li><li> Preparare il vettore di espressione pMalc2x digerendo 2 ug di questo vettore con ciascuna 1 ml BamHI (20.000 U / ml) e NotI (10.000 U / ml) enzimi di restrizione per 1 ora a 37 ° C. Purificare il vettore da un gel di agarosio come descritto sopra (1.3). Nota: Entrambi i campioni (1.2 e 1.4) possono essere conservati a -20 ° C. </li><li> Legare l&#39;inserto purificato nel vettore pMalc2x digerito nei siti di restrizione BamHI e NotI con 1 ml T4-DNA ligasi (400.000 U / ml) per 15 minuti a temperatura ambiente (RT) (Figura 2B e 2C). Utilizzare almeno 20 ng del vettore pMalc2x e un vettore: rapporto inserto 1: 3 (peso molecolare). La miscela di legamento è di solito ~ 20 l. </li><li> Transform 2 ul di miscela di ligasi in E. 50 microlitri chimicamente competenti coli DH5α cellule batteriche utilizzando un protocollo standard di trasformazione (10 min in ghiaccio, heat shock 45 sec a 42 ° C) e crescere in 600media SOC ml per 1 ora a 37 ° C. Centrifugare le cellule trasformate per 3 minuti a 1000 xg, scartare il surnatante e risospendere il pellet in 30 ml di mezzo SOC. <ol><li> Piatto mix trasformazione su piastre LB-Agar, contenente 100 ug / ml di ampicillina per la selezione, e crescere durante la notte a 37 ° C. Selezionare almeno cinque colonie. </li></ol></li><li> Per ciascuna delle cinque colonie, inoculare 2-3 coltura ml di LB-medium supplementato con 50 ug / ml di ampicillina (LB-amp) e crescere agitando per una notte a 160 rpm a 37 ° C. </li><li> Estrarre i plasmidi dalla cultura durante la notte utilizzando un kit commerciale. Verificare la presenza dell&#39;inserto dominio P mediante sequenziamento con un primer forward pMalc2x (5&#39;-TCAGACTGTCGATGAAGC-3 &#39;) e primer reverse (5&#39;-GATGTGCTGCAAGGCGAT-3&#39;). </li></ol> Espressione 2. P Domain <ol><li> Trasforma 1 ml (150 ng / ml - 400 ng / ml) della codifica vettore pMalc2x per il MBP-His-P dominioproteina di fusione in 50 ml di E. competente cellule coli BL21 utilizzando un protocollo standard di trasformazione (10 min in ghiaccio, heat shock 45 sec a 42 ° C) e crescono in media SOC 600 ml per 1 ora a 37 ° C. Subculture in 120 ml di LB-amp durante la notte a 160 giri al minuto e 37 ° C. </li><li> Seminare nove litri (ad esempio, 6 x 5 L palloni con 1,5 L di medie ciascuna) di LB-amp con la sottocultura (1: 100). Crescere le cellule agitando a 160 giri al minuto e 37 ° C fino a quando la OD 600 raggiunge 0,4-0,6. Successivamente, abbassare la temperatura a 22 ° C per ~ 1 ora e quindi indurre l&#39;espressione di proteine ​​con 0,66 mM di isopropil-β-D-thiogalactopyranoside (IPTG) 8. Crescere le cellule durante la notte a 22 ° C (~ 18 ore). Nota: La temperatura può essere variata, ma si consiglia di utilizzare 22 ° C o inferiore. </li><li> Raccogliere le cellule per centrifugazione (10.543 xg, 15 min, 4 ° C). Eliminare il surnatante e congelare il pellet cellulare a -20 ° C. </li></ol>° Purificazione Step e proteasi fenditura <ol><li> Preparare buffer che vengono utilizzati durante le fasi di purificazione proteine ​​da soluzioni madre di garantire riproducibilità e stabilità degli esperimenti. Preparare quattro tamponi differenti per il metallo immobilizzato ionico cromatografia di affinità (IMAC), ciascuno contenente una diversa concentrazione di imidazolo (10 mM, 20 mM, 50 mM e 250 mM). Per la SEC, preparare un tampone di gel-filtrazione (GFB) con una concentrazione di sale più elevata, ma senza imidazolo. Utilizzare acqua deionizzata e filtrare tutti i buffer prima di utilizzarlo con una dimensione dei pori di 0,45 micron. Nota: Per una dettagliata schema di preparazione del buffer, fare riferimento alla Tabella 1. </li><li> Scongelare il pellet di cellule dalla cultura nine litri e sciogliere in 150 ml di PBS a 4 ° C. Sonicare la sospensione cellulare tre volte per 2 min (potenza 130 W, ampiezza 20%, frequenza impulsi 50%) per distruggere le cellule. Mantenere la sospensione cellulare in ghiaccio durante sonicazione. </li><li> Centrifuge la sospensione cellulare sonicato (43.667 xg, 30 min, 4 ° C) per separare detriti cellulari dal supernatante contenente proteina espressa. Raccogliere il surnatante e scartare il pellet. </li><li> Lavare ed equilibrare 10 ml (= 1 volume colonna [CV]) impasto di Nichel (Ni) perline agarosio -nta con 10 mM tampone imidazolo in una colonna cromatografia. Aggiungere le perline Ni equilibrati per il surnatante dal punto 3.3 contenente espresso MBP-His-P dominio proteina di fusione e incubare per 30 minuti a 4 ° C con rotazione lenta. </li><li> Dopo l&#39;incubazione, applicare l&#39;intera miscela Ni-bead-proteina ad una colonna cromatografica. Lavare la colonna lentamente con ogni 5 CV di 10 mm, 20 mm, e 50 mm buffer imidazolo, a partire da 10 mm, 20 mm e poi le 50 mm (Figura 3A). </li><li> Eluire il MBP-His-P proteina di fusione del dominio utilizzando 250 mm tampone imidazolo (Figura 3A). Durante l&#39;eluizione, controlla l&#39;280nm OD per verificare l&#39;eluizione della proteina di fusione (l&#39;aumento OD 280nm). Continuare fino a quando la eluizione 280nm OD scende a ~ 0.1. Lavare le perline con quantità eccessive di 250 mm tampone imidazolo (almeno 10 CV), seguita da almeno 10 CV di 10 mm tampone imidazolo. Salvare le perline per la seconda fase di purificazione (sezione 4). </li><li> Verificare la presenza della proteina di fusione del dominio MBP-His-P con SDS-PAGE su gel 12% SDS-poliacrilammide (10 x 8 cm) 9 (Figura 3A). Eseguire gel elettroforesi a 45 A e 200 V per 45 min. </li><li> Concentrato (ad esempio, utilizzando un concentratore commerciale) dominio MBP-His-P proteina di fusione eluita ad una concentrazione finale di ~ 3 mg / ml. Cleave la fusione del dominio MBP-His-P con HRV-3C proteasi durante la dialisi contro 2 L di 10 mM di tampone imidazolo (~ 1: 100) notte a 4 ° C (Figura 3A). A seconda del volume finale di proteine ​​concentrate, eseguire la dialisi in una cassetta dialisi o tubo di dialisi. Nota: La quantità di HRV-3C proteasi per le proteineclivaggio viene calcolato secondo la specifica attività della proteasi (2 U / ml, 1 U è sufficiente per fendere 100 mg di proteina) e la quantità di proteina di fusione eluite che varia il livello di espressione e può essere stimata dal risultato SDS-PAGE (3.7 ). </li></ol> 4. 2 ° Fase Purificazione <ol><li> Equilibrare le Ni-perle dal punto 3.6 a 10 tampone imidazolo mm. </li><li> Incubare la proteina dializzata dal punto 3.7 (contenente dominio spaccati P, proteine ​​MBP e HRV-proteasi) con le Ni-perle equilibrate (4.1) per 30 minuti a 4 ° C con rotazione lenta. </li><li> Applicare la miscela Ni-bead a una colonna e raccogliere il flow-through (dominio P spaccati) (Figura 3B). Misurare la concentrazione di proteine ​​come viene fuori la colonna fino alla 280nm OD raggiunge ~ 0.1. Nota: Il MBP-His dovrebbe rimanere legato alle Ni-perline (figura 3b). </li><li> Verificare la presenza di dominio spaccati P mediante SDS-PAGE con un 1Gel 2% come sopra descritto (Figura 3B). Concentrare il dominio P eluita a ~ 3 mg / ml e dializzare notte a 4 ° C contro GFB per successiva purificazione SEC. </li></ol> 5. 3 ° Purificazione Passo <ol><li> Le pompe di lavaggio e tubi del sistema di purificazione HPLC e pre-equilibrare la SEC-colonna (vedi elenco Materials) con GFB. </li><li> Iniettare il dominio P per la colonna ad una portata di 1 ml / min usando un Superloop (fino a 12 ml) o loop (fino a 3 ml), a seconda del volume del campione concentrato. Dopo l&#39;iniezione è terminata, aumentare la portata di 2,5 ml / min. </li><li> Come le OD aumenta e il dominio P si stacca la colonna, raccogliere frazioni di 1,5 ml. Controllare frazioni utilizzando SDS-PAGE con un gel al 12% (Figura 4A e 4B). Pool solo i più puri frazioni e concentrarsi per ~ 3 mg / ml e ~ 8 mg / ml. Nota: dopo ~ 110 ml (volume vuoto) la maggior parte delle impurità vengono eluite da una colonna SEC con un volume di 320 ml letto. Ildominio P è generalmente eluito come dimero. Elution tempo / volume del dimero dominio P dipende dal grado di preparazione (pg) della colonna SEC. </li></ol> 6. Cristallizzazione della P Domain <ol><li> Utilizzare il dominio P a ~ 3 mg / ml e 8 mg / ml per lo screening cristallizzazione iniziale. Preparare almeno 100 ml di dominio P per la concentrazione per gli schermi iniziali con 384 condizioni di screening disponibili in commercio. Eseguire lo screening a 18 ° C in un formato piastra a 96 pozzetti, dove il serbatoio contiene 100 ml di soluzione madre e una goccia è composta da 0,2 soluzione madre microlitri e 0,2 microlitri proteina. </li><li> Ripetere e ottimizzare le condizioni di cristallizzazione di successo. Pertanto, utilizzare piastre 15 pozzetti contenenti 3 righe. Impostare la prima fila con soluzione madre 100%, la seconda fila con soluzione al 90% madre e 10% di acqua, e la terza fila con soluzione madre 80% e 20% di acqua. Utilizzare una dimensione goccia di 2 ml (soluzione madre di proteine ​​+ 1 ml 1 ml) e 50081; l di soluzione madre come volume del serbatoio. </li><li> Utilizzare le condizioni di cristallo ottimizzate per la co-cristallizzare il dominio P con leganti. Preparare piastre come descritto al punto 6.2. Invece di 2 microlitri dimensioni di goccia, impostare gocce contenenti 1 ml soluzione madre, 1 proteine ​​ml e 1 ml di ligando ad una concentrazione di 1 mg / ml. </li><li> Raccogliere set di dati di singoli cristalli utilizzando radiazione di sincrotrone. Eseguire la sostituzione molecolare utilizzando strutture di dominio P pubblicati con una somiglianza alta sequenza iniziale di ricerca del modello 6,10-13. Nota: La presenza di un ligando si presenta come non-modellato blob di densità elettronica. </li></ol>

<ol>
	<li>Ahmed, S. 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=Global+prevalence+of+norovirus+in+cases+of+gastroenteritis:+a+systematic+review+and+meta-analysis.">Global prevalence of norovirus in cases of gastroenteritis: a systematic review and meta-analysis.</a> Lancet Infect. Dis. 14, 725-730 (2014).</li><li>Prasad, B. V., Matson, D. O., Smith, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+structure+of+calicivirus.">Three-dimensional structure of calicivirus.</a> J. Mol. Biol. 240, 256-264 (1994).</li><li>Choi, J. M., Hutson, A. M., Estes, M. K., Prasad, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+resolution+structural+characterization+of+recognition+of+histo-blood+group+antigens+by+Norwalk+virus.">Atomic resolution structural characterization of recognition of histo-blood group antigens by Norwalk virus.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 9175-9180 (2008).</li><li>Marionneau, S., 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=Norwalk+virus+binds+to+histo-blood+group+antigens+present+on+gastroduodenal+epithelial+cells+of+secretor+individuals.">Norwalk virus binds to histo-blood group antigens present on gastroduodenal epithelial cells of secretor individuals.</a> Gastroenterology. 122, 1967-1977 (2002).</li><li>Jones, M. K., 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=Enteric+bacteria+promote+human+and+mouse+norovirus+infection+of+B+cells.">Enteric bacteria promote human and mouse norovirus infection of B cells.</a> Science. 346, 755-759 (2014).</li><li>Hansman, G. S., 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=Crystal+structures+of+GII.10+and+GII.12+norovirus+protruding+domains+in+complex+with+histo-blood+group+antigens+reveal+details+for+a+potential+site+of+vulnerability.">Crystal structures of GII.10 and GII.12 norovirus protruding domains in complex with histo-blood group antigens reveal details for a potential site of vulnerability.</a> J. Virol. 85, 6687-6701 (2011).</li><li>Fath, S., 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=Multiparameter+RNA+and+codon+optimization:+a+standardized+tool+to+assess+and+enhance+autologous+mammalian+gene+expression.">Multiparameter RNA and codon optimization: a standardized tool to assess and enhance autologous mammalian gene expression.</a> PLoS One. 6 (e17596), (2011).</li><li>Jacob, F., Monod, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+regulatory+mechanisms+in+the+synthesis+of+proteins.">Genetic regulatory mechanisms in the synthesis of proteins.</a> J. Mol. Biol. 3, 318-356 (1961).</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, 680-685 (1970).</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=Automatic+Processing+of+Rotation+Diffraction+Data+from+Crystals+of+Initially+Unknown+Symmetry+and+Cell+Constants.">Automatic Processing of Rotation Diffraction Data from Crystals of Initially Unknown Symmetry and Cell Constants.</a> J. Appl. Crystallogr. 26, 795-800 (1993).</li><li>Mccoy, A. 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=Phaser+crystallographic+software.">Phaser crystallographic software.</a> J. Appl. Crystallogr. 40, 658-674 (2007).</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. Sect. D-Biol. Crystallogr. 66, 486-501 (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. Sect. D-Biol. Crystallogr. 66, 213-221 (2010).</li><li>Koromyslova, A. D., Hansman, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanobody+binding+to+a+conserved+epitope+promotes+norovirus+particle+disassembly.">Nanobody binding to a conserved epitope promotes norovirus particle disassembly.</a> J. Virol. 89, 2718-2730 (2015).</li><li>Hansman, G. S., 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+basis+for+broad+detection+of+genogroup+II+noroviruses+by+a+monoclonal+antibody+that+binds+to+a+site+occluded+in+the+viral+particle.">Structural basis for broad detection of genogroup II noroviruses by a monoclonal antibody that binds to a site occluded in the viral particle.</a> J. Virol. 86, 3635-3646 (2012).</li><li>Singh, B. K., Leuthold, M. M., Hansman, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+noroviruses'+fondness+for+histo-blood+group+antigens.">Human noroviruses' fondness for histo-blood group antigens.</a> J. Virol. 89, 2024-2040 (2015).</li><li>Leuthold, M. M., Dalton, K. P., Hansman, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+analysis+of+a+rabbit+hemorrhagic+disease+virus+binding+to+histo-blood+group+antigens.">Structural analysis of a rabbit hemorrhagic disease virus binding to histo-blood group antigens.</a> J. Virol. 89, 2378-2387 (2015).</li></ol>

The authors have nothing to disclose.

Qui, descriviamo un protocollo per l&#39;espressione e la purificazione di domini P norovirus in alta qualità e quantità. Norovirus non sono ben studiati e dati strutturali sono continuamente necessari. A nostra conoscenza, la produzione dominio P utilizzando altri protocolli (ad esempio, domini P GST-tagged) è stato problematico, fino ad ora, e dati strutturali sufficienti interazione norovirus-host sono stati dispersi. Con il metodo qui descritto, abbiamo recentemente contribuito in modo significativo alla...

Norovirus umani sono la principale causa di gastroenterite acuta in tutto il mondo 1. Questi virus appartengono alla famiglia Caliciviridae, di cui ci sono almeno cinque generi, tra cui Norovirus, Sapovirus, Lagovirus, Vesivirus, e Nebovirus. Nonostante il loro elevato impatto sul sistema sanitario e ampia distribuzione, lo studio dei norovirus umani è ostacolata dalla mancanza di un sistema di coltura cellulare robusto. Fino ad oggi, non ci sono vaccini autorizzati o strategie antivirali disponibili. La proteina capside maggiore norovirus, definito VP1, può essere diviso in un guscio (S) e un dominio sporgente (P) del dominio 2. Il dominio P è collegato al dominio S da una regione cerniera flessibile (H). Il dominio S forma una impalcatura attorno alla RNA virale, mentre il dominio P costituisce la parte più esterno del capside virale. Il dominio P assembla in dimeri biologicamente rilevanti quando espresso in batteri. Il P dIMER interagisce con le strutture di carboidrati, denominati antigeni del gruppo isto-sangue (HBGAs) che sono presenti come antigeni solubili nella saliva e trovato su alcune cellule ospiti 3. L&#39;interazione dominio HBGA P è pensato per essere importante per l&#39;infezione 4. Infatti, un recente rapporto ha rivelato l&#39;importanza di HBGAs sintetici o HBGA esprimono batteri per l&#39;infezione da norovirus umano in vitro 5. Gli studi in corso per quanto riguarda l&#39;attaccamento cellula ospite di norovirus sono principalmente eseguiti con particelle virus-simili (VLP) che possono essere espresse in cellule di insetto o con i domini P ricombinanti espressi in Escherichia coli (E. coli). Per capire le interazioni P dominio HBGA a risoluzione atomica, strutture complesse P dominio HBGA possono essere risolti mediante cristallografia a raggi X. Qui, descriviamo un protocollo per l&#39;espressione del dominio P e purificazione che permette la produzione di dominio P in alta quantità e qualità da utilizzare per crystall raggi Xografia. Inoltre, questo metodo può essere applicato per altri domini calicivirus P e proteine ​​non strutturali. Il dominio P è codone-ottimizzato per E. espressione coli e clonato in un vettore di trasferimento standard. Il dominio P viene poi ri-clonato in un vettore di espressione che codifica una polyhistidine tag (His) e una proteina legante mannosio (MBP) che sono seguiti da un sito proteasi clivaggio. Il-His-P MBP proteina di fusione del dominio è espresso in E. coli, seguita da tre fasi di purificazione. La proteina di fusione del dominio MBP-His-P viene purificato mediante cromatografia immobilizzato metallo ionica affinità (IMAC). Successivamente, la proteina di fusione viene scisso con rinovirus umano (HRV) proteasi -3C e il dominio P è separato dal MBP-His da un ulteriore passaggio di purificazione IMAC. Infine, il dominio P viene purificato mediante cromatografia di esclusione dimensionale (SEC). Il dominio P purificato può essere utilizzato per la cristallografia a raggi X. Proiezione delle condizioni di cristallizzazione delle proteine ​​è perfoconfermate con i kit di screening disponibili in commercio che utilizzano differenti concentrazioni di proteine ​​dominio P. crescita dei cristalli si osserva e le condizioni più promettenti sono ottimizzate. Con i metodi qui descritti, è possibile passare dal gene alla proteina di strutturare in meno di quattro settimane. Pertanto, il nostro metodo di espressione P dominio, purificazione e cristallizzazione è adatto per studiare l&#39;interazione norovirus-ospite a livello molecolare e di fornire dati importanti per aiutare nella progettazione e farmaci di screening vaccino up-to-date.

<table>
	<tbody>
		<tr>
			<td>P domain DNA</td>
			<td>Life Technologies</td>
			<td></td>
			<td>GeneArt Gene Synthesis</td>
		</tr>
		<tr>
			<td>pMalc2x&#160; vector</td>
			<td>On request</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BamHI</td>
			<td>New England Biolabs</td>
			<td>R0136L</td>
			<td></td>
		</tr>
		<tr>
			<td>NotI</td>
			<td>New England Biolabs</td>
			<td>R0189L</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>New England Biolabs</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td></td>
		</tr>
		<tr>
			<td>S.O.C. Medium</td>
			<td>Life Technologies</td>
			<td>15544-034</td>
			<td></td>
		</tr>
		<tr>
			<td>Econo-Column Chromatography Column</td>
			<td>Bio-Rad</td>
			<td>7372512</td>
			<td>2.5 cm x 10 cm, possible to use other size</td>
		</tr>
		<tr>
			<td>Ni-NTA Agarose</td>
			<td>Qiagen</td>
			<td>30210</td>
			<td></td>
		</tr>
		<tr>
			<td>Vivaspin 20</td>
			<td>GE Healthcare</td>
			<td>various</td>
			<td>cutoff of 10 kDa, 30 kDa and 50 kDa used</td>
		</tr>
		<tr>
			<td>Subcloning Efficiency DH5&#945; Competent Cells</td>
			<td>Life Technologies</td>
			<td>18265-017</td>
			<td></td>
		</tr>
		<tr>
			<td>One Shot BL21(DE3) Chemically Competent E. coli</td>
			<td>Life Technologies</td>
			<td>C6000-03</td>
			<td></td>
		</tr>
		<tr>
			<td>HRV 3C Protease</td>
			<td>Merck Millipore</td>
			<td>71493</td>
			<td></td>
		</tr>
		<tr>
			<td>HiLoad 26/600 Superdex 75 PG</td>
			<td>GE Healthcare</td>
			<td>28-9893-34</td>
			<td>SEC column</td>
		</tr>
		<tr>
			<td>JCSG Core suites</td>
			<td>Qiagen</td>
			<td>various</td>
			<td>4 screens with each 96 wells</td>
		</tr>
		<tr>
			<td>Carbohydrates</td>
			<td>Dextra Laboratories, UK</td>
			<td>various</td>
			<td>Blood group products</td>
		</tr>
	</tbody>
</table>

production of human norovirus protruding domains in e. coli for x-ray crystallography

Il capside norovirus è composto da una singola proteina strutturale principale, denominato VP1. VP1 è suddiviso in un guscio (S) dominio e un dominio sporgenti (P). Il dominio S forma una impalcatura contigua intorno l&#39;RNA virale, mentre le forme di dominio P picchi virali sul dominio S e contiene determinanti per antigenicità e host-cellule interazioni. Il dominio P lega strutture di carboidrati, ad esempio., Antigeni del gruppo isto-sangue, che si pensa siano importanti per le infezioni norovirus. In questo protocollo, si descrive un metodo per la produzione di domini norovirus P di alta qualità con rese elevate. Queste proteine ​​possono quindi essere utilizzati per la cristallografia a raggi X ed ELISA per studiare antigenicità e host-cellule interazioni. Il dominio P viene prima clonato in un vettore di espressione e quindi espressa in batteri. La proteina è purificata utilizzando tre fasi che coinvolgono immobilizzati metallo-ioni cromatografia di affinità e le dimensioni cromatografia di esclusione. Inlinea di principio, è possibile clonare, espresso, purificare, e cristallizzare le proteine ​​in meno di quattro settimane, il che rende questo protocollo un sistema rapido per l&#39;analisi dei ceppi di norovirus emergenti.

Lo schema del protocollo descritto è illustrato in Figura 1. Il protocollo comprende 6 parti principali che includono la clonazione del gene bersaglio, espressione, una purificazione tre fasi, e la cristallizzazione. Figura 2 illustra il disegno del costrutto di espressione (CE) e caratteristiche del vettore di espressione pMalc2x. La sequenza del sito di clonaggio multiplo (MCS) del vettore pMalc2x mostra restrizione e proteasi siti di taglio. ...

Watch this Scientific Journal Video about Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography at JoVE.com

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

Produzione di Norovirus umani sporgenti Domini in<em&gt; E. coli</em&gt; Per la cristallografia a raggi X

Qui, descriviamo un metodo per esprimere e purificare norovirus di alta qualità sporgenti (P) domini in E. coli per l&#39;utilizzo in studi di cristallografia a raggi X. Questo metodo può essere applicato ad altri domini calicivirus P, così come le proteine ​​non-strutturali, cioè., Proteina virale genoma-linked (VPG), proteasi, e RNA RNA dipendente polimerasi (RdRp).

The norovirus capsid is composed of a single major structural protein, termed VP1. VP1 is subdivided into a shell (S) domain and a protruding (P) domain. ...

production-human-norovirus-protruding-domains-e-coli-for-x-ray

Research

JoVE Journal

Biology

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

8.6K Views.  University of Heidelberg and the German Cancer Research Center.  Qui, descriviamo un metodo per esprimere e purificare norovirus di alta qualità sporgenti (P) domini in E. coli per l'utilizzo in studi di cristallografia a raggi X. Questo metodo può essere applicato ad altri domini calicivirus P, così come le proteine ​​non-strutturali, cioè., Proteina virale genoma-linked (VPG), proteasi, e RNA RNA dipendente polimerasi (RdRp). 

Video: Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42&deg;C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37&deg;C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.

Trasformazione del DNA plasmidi in E. coli con il metodo Heat Shock

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign ...

Ricerca

Biologia

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning.
We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.

We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows the reconstruction of internal structures in their natural state without the artefacts produced by histological sectioning.

Non invasive di visualizzazione 3D con sub-micron Risoluzione Utilizzando sincrotrone a raggi-X, la tomografia

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard ...

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types and is widely believed to be directly associated with the development of cancer in humans. DNA damage, such as that induced by UV irradiation, severely impairs the ability of replication to duplicate the genomic template accurately. A number of gene products have been identified that are required when replication encounters DNA lesions in the template. However, a remaining challenge has been to determine how these proteins process lesions during replication in vivo. Using Escherichia coli as a model system, we describe a procedure in which two-dimensional agarose-gel analysis can be used to identify the structural intermediates that arise on replicating plasmids in vivo following UV-induced DNA damage. This procedure has been used to demonstrate that replication forks blocked by UV-induced damage undergo a transient reversal that is stabilized by RecA and several gene products associated with the RecF pathway. The technique demonstrates that these replication intermediates are maintained until a time that correlates with the removal of the lesions by nucleotide excision repair and replication resumes.

Visualization of UV-induced Replication Intermediates in E. coli using Two-dimensional Agarose-gel Analysis

We present a procedure by which two-dimensional agarose-gel analysis can be used to identify the structure of replication intermediates that occur following UV irradiation.

Visualizzazione dei raggi UV-indotta Intermedi replica in E. coli Utilizzando bidimensionale agarosio-gel Analisi

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types ...

Protein Crystallization for X-ray Crystallography

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The availability of atomic resolution structures provides a deep and unique understanding of protein function, and helps to unravel the inner workings of the living cell. To date, 86% of the Protein Data Bank (rcsb-PDB) entries are macromolecular structures that were determined using X-ray crystallography.
To obtain crystals suitable for crystallographic studies, the macromolecule (e.g. protein, nucleic acid, protein-protein complex or protein-nucleic acid complex) must be purified to homogeneity, or as close as possible to homogeneity. The homogeneity of the preparation is a key factor in obtaining crystals that diffract to high resolution (Bergfors, 1999; McPherson, 1999).
Crystallization requires bringing the macromolecule to supersaturation. The sample should therefore be concentrated to the highest possible concentration without causing aggregation or precipitation of the macromolecule (usually 2-50 mg/ mL). Introducing the sample to precipitating agent can promote the nucleation of protein crystals in the solution, which can result in large three-dimensional crystals growing from the solution. There are two main techniques to obtain crystals: vapor diffusion and batch crystallization. In vapor diffusion, a drop containing a mixture of precipitant and protein solutions is sealed in a chamber with pure precipitant. Water vapor then diffuses out of the drop until the osmolarity of the drop and the precipitant are equal (Figure 1A). The dehydration of the drop causes a slow concentration of both protein and precipitant until equilibrium is achieved, ideally in the crystal nucleation zone of the phase diagram. The batch method relies on bringing the protein directly into the nucleation zone by mixing protein with the appropriate amount of precipitant (Figure 1B). This method is usually performed under a paraffin/mineral oil mixture to prevent the diffusion of water out of the drop.
Here we will demonstrate two kinds of experimental setup for vapor diffusion, hanging drop and sitting drop, in addition to batch crystallization under oil.

The 3-D structure of a molecule provides a unique understanding of how the molecule functions. The principal method for structure determination at near-atomic resolution is X-ray crystallography. Here, we demonstrate the current methods for obtaining three-dimensional crystals of any given macromolecule that are suitable for structure determination by X-ray crystallography.

Cristallizzazione delle proteine ​​per la cristallografia a raggi X

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The ...

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

Protocolli di selezione mutagenesi e funzionale per l&#39;evoluzione Regia di proteine ​​in E. coli</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

Una piattaforma espressione conveniente e generale per la produzione di proteine ​​secrete da cellule umane

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

3D Printing of Preclinical X-ray Computed Tomographic Data Sets

Three-dimensional printing allows for the production of highly detailed objects through a process known as additive manufacturing. Traditional, mold-injection methods to create models or parts have several limitations, the most important of which is a difficulty in making highly complex products in a timely, cost-effective manner.1 However, gradual improvements in three-dimensional printing technology have resulted in both high-end and economy instruments that are now available for the facile production of customized models.2 These printers have the ability to extrude high-resolution objects with enough detail to accurately represent in vivo images generated from a preclinical X-ray CT scanner. With proper data collection, surface rendering, and stereolithographic editing, it is now possible and inexpensive to rapidly produce detailed skeletal and soft tissue structures from X-ray CT data. Even in the early stages of development, the anatomical models produced by three-dimensional printing appeal to both educators and researchers who can utilize the technology to improve visualization proficiency. 3, 4 The real benefits of this method result from the tangible experience a researcher can have with data that cannot be adequately conveyed through a computer screen. The translation of pre-clinical 3D data to a physical object that is an exact copy of the test subject is a powerful tool for visualization and communication, especially for relating imaging research to students, or those in other fields. Here, we provide a detailed method for printing plastic models of bone and organ structures derived from X-ray CT scans utilizing an Albira X-ray CT system in conjunction with PMOD, ImageJ, Meshlab, Netfabb, and ReplicatorG software packages.

Using modern plastic extrusion and printing technologies, it is now possible to quickly and inexpensively produce physical models of X-ray CT data taken in a laboratory. The three -dimensional printing of tomographic data is a powerful visualization, research, and educational tool that may now be accessed by the preclinical imaging community.

La stampa 3D di preclinica X-tomografia computerizzata a raggi Data Set

Three-dimensional printing allows for the production of highly detailed objects through a process known as additive manufacturing.  Traditional, ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Produzione efficiente e purificazione di ricombinante murino kindlina-3 da cellule di insetto per Biofisica Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Green Fluorescent Protein-base Espressione Proiezione di proteine ​​di membrana in<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

We describe a method to knock down gene expression in a growing population of E. coli cells using sequence-targeted sRNA expression cassettes delivered by an M13 phagemid vector.