Noi riconosciamo il dottor Amir Aharoni per il suo sostegno e Gheula Davidov, Noam Grimberg e Chen Guttman per i loro consigli e commenti.

1. Clonazione ed espressione di Mama Gene in E. coli Il gene mutante mamAΔ41 è stato amplificato utilizzando la reazione a catena della polimerasi (PCR) dal DNA genomico di Magnetospirillum magneticum AMB-1, con primer: 5&#39;-GCATTACGCATATGGACGACATCCGCCAGGTG-3 &#39;e 5&#39;-GCGCGGCAGCCATA-TGGCATACG-3&#39;. Nei frammenti di DNA amplificato, un sito che NcoI è stato introdotto al codone di inizio ATG e il codone di terminazione è stata sostituita con un sito ScoI. I frammenti sono stati digeriti con NcoI e Saci e clonato nei rispettivi siti di pET52b (+), dando luogo a pET52bMamAΔ41-AMB1. In questo costrutto, il gene è stato fuso mamAΔ41 in-frame con il suo tag-10 al C-terminale. Il plasmide è stato elettroporate in ceppo di E. coli BL21. E. coli ceppo BL21 ospitare pET52bMamAΔ41 è stato coltivato in auto-induzione (15) su terreno contenente ampicillina (50 mg / ml) 310 ° K per 3 ore. La temperatura di coltivazione è stato poi spostato da 310 ° a 300 ° K e mantenuti per ulteriori 48 ore a 300 ° K. Le cellule sono state raccolte mediante centrifugazione a 5465g per 10 min a 277 ° K. 8 litri cultura prodotta 60 grammi di pellet umido. 2. Bioinformatica Calcoli I calcoli di peso molecolare (MW), secondo la sequenza aminoacidi acidi e l&#39;assorbimento previsto di 1mg/1ml di proteine ​​a 280 nm, utilizzando il server ProtParam (http://www.expasy.ch/tools/protparam.html). Per 10His-Tag-MamAΔ41 il MW è 22.529 Da (240 aminoacidi) e per MamAΔ41 (con 9 aminoacidi sinistra dopo la sua rimozione dal tag-trombina) il Mw è 20596.5Da (187 aminoacidi). L&#39;assorbimento del predetto 1mg/1ml di proteine ​​a 280 nm per 10His-Tag-MamAΔ41 è 0,595 e per MamAΔ41 è 0,579. Inoltre, la sequenza di amminoacidi non contiene residui di cisteina. Pertanto, agenti riducenti non sono richiesti durante il processo di purificazione. 3. Purificazione di MamAΔ41 <ol><li> Per la preparazione delle soluzioni madre, pesare e preparare (200 ml) nelle concentrazioni seguenti: imidazolo (MW 228,2 g / mol) 4M soluzione NaCl (Mw 58,44 g / mol) 5M soluzione, Tris-HCl (Mw 121,3 g / mol soluzione) 1M, aggiustare il pH = 8. Aggiungere acqua bidistillata (DDW) e miscelare con un vortice fino a quando la soluzione è limpida. Soluzioni di filtraggio con filtro 0.22μm e conservare a temperatura ambiente. </li><li> Preparare e filtro 6 tamponi freschi soluzioni di riserva (Tabella 1). </li><li> Diluire e sospendere le cellule in un tampone di A in rapporto 1:2, grammi di cellule per tamponare volume (ml). </li><li> Aggiungere 10μl per ogni 10gr di batteri di DNasi I (1 mg / ml) al fine di rompere frammenti di DNA nel campione. Aggiungere 1ml per ogni 20gr di batteri di cocktail EDTA inibitore della proteasi libero. Incubare per 20 minuti sul ghiaccio. </li><li> Turbativa celle da due cicli di stampa francese a 25.000 psi. A differenza di sonicazione, la stampa francese si rivolge ad alto volume (10 ml&gt;) estrusione in cella forzando le cellule ad alta pressione attraverso un orifizio sottile. Il pistone deve essere raffreddato in ghiaccio per dieci minuti prima del suo utilizzo e il montaggio a causa di accumulo di temperatura. </li><li> Trasferire le cellule lisate in provette da 25 ml ultracentrifuga ed equilibrio con precisione (50mg) con tampone A fino a quando l&#39;esemplare ha raggiunto la linea del collo del tubo s. </li><li> Detriti cellulari Ultra separata per centrifugazione a 270.000 g per 1 ora a 277 ° K. </li><li> Preparare una casa colonna gravità Ni NTA (2,5 cm di diametro) con l&#39;aggiunta di 4 ml di Ni NTA resina, che si conserva in etanolo al 20%, sulla colonna. Lasciar cadere del tutto fluido, quindi lavare con 40 ml di DDW e lasciar cadere del tutto fluido. Pre-equilibrata la resina nella colonna con 40 ml di tampone A. </li><li> Applicare la frazione solubile da ultra-centrifugazione tubi sulla colonna gravità Ni NTA. Lasciar cadere liquido, raccogliere flow-through (non legato) e conservare a 277K. </li><li> Raccogliere campioni di pellet da ultra-centrifugazione tubi utilizzando una piccola mancia per la demolizione del pellet. Mescolare la punta in 50μl del 5% β-mercaptoetanolo in 2X SDS-PAGE tampone del campione. </li><li> Lavare la colonna con 100 ml di tampone B. Lasciate che la goccia fluida, raccogliere flow-through e preservare a 277K. </li><li> Preparare 15 segnato tubi eppendorf (1,5 ml). </li><li> Eluizione - valvola colonna stretta e quindi caricare 1 ml di tampone C nella colonna; incubare per 3 minuti seguito da apertura della valvola e di flusso attraverso la raccolta. Ripetere questa operazione tre volte. </li><li> Resto eluire di sospensione a incrementi di 1 ml di tampone C e chiudere la valvola. </li><li> Misura OD a 280 nm delle frazioni e di valutare la localizzazione del picco proteine, se l&#39;OD è ancora&gt; 0,3, campioni supplementari sono necessari. </li><li> SDS-PAGE analisi delle frazioni raccolte. Tale analisi deve essere eseguita anche con gli esempi seguenti, le proteine ​​non legato flow-through, lavare flow-through e uLTRA-pellet centrifugazione del campione. Mescolare 10μl di ciascun campione con 10μl del 5% β-mercaptoetanolo in 2X SDS-PAGE tampone del campione. Caricare i campioni, il 15% SDS-PAGE e correre per 45 minuti a 180 Volt. </li><li> Macchia il gel in 15 ml di soluzione InstantBlue, agitare per 1 ora. Assicurarsi che la scatola di plastica è coperto e protetto dalla luce. </li><li> Valutare gel, se espressione della proteina è alta e specifiche in base alle fasce indicative, unione selezionato frazioni eluite. </li><li> Al fine di rimuovere 10-His-tag, aggiungere trombina di origine bovina (10 unità / mL), 10μl trombina per 1 mg di proteina, secondo le indicazioni del produttore per il campione combinato. </li><li> La dialisi è utilizzato per eccesso di rimozione imidazolo e diminuire le concentrazioni di NaCl per i livelli necessari durante l&#39;associazione a scambio ionico. Per dialisi, lunghezza desiderata di 7 kD peso molecolare tagliato fuori (MWCO) tubi di dialisi. Lavare con DDW e fare un nodo ad una estremità (o sigillo con clips). Trasferire la soluzione proteina fusa sulla borsa lasciando qualche spazio in alto e morsetto in su. </li><li> Immergere il tubo di dialisi in 4 buffer di dialisi litri refrigerato (Buffer D). Agitare la soluzione lentamente durante la notte a 277 ° K. </li><li> Estrarre il tubo di dialisi del bicchiere, fare un taglio piccolo e trasferire la proteina a tubo 50ml. </li><li> Cromatografia a scambio ionico: assemblare preconfezionati MonoQ 4.6/100 colonna PE sul veloce Performance Liquid Chromatography (FPLC). Lavare la colonna con 5 volumi di colonna (CV) di DDW acqua filtrata. Equilibrata la colonna con 5 CV di tampone D. </li><li> Lavare il ciclo di iniezione con DDW (due volumi loop) e ripetere con il tampone D. carico della proteina nel ciclo di iniezione. </li><li> Iniettare il campione (flusso di 1 ml / min) e iniziare a raccogliere frazioni per SDS-PAGE rilevazione di proteine ​​senza limiti. </li><li> Se il volume delle proteine ​​iniziale (passo 3.23) è più grande ansa di iniezione, ripetere i passaggi 3,25-3,26, senza lavare il ciclo, fino a quando l&#39;intero campione è stato caricato su colonna. </li><li> Lavare la colonna con 3 CV di tampone C al fine di rimuovere le proteine ​​illimitato. </li><li> Eluire la proteina con un gradiente di 60 min lineare di 40 2000mm NaCl tra i respingenti D &amp; E (quando E tampone al 100% è la fine del gradiente), portata 1 ml / min (anche se portate superiori può essere utilizzato nel costo del volumi più grandi, ma meno concentrati, i picchi delle proteine). Raccogliere i picchi proteine ​​come appaiono nel cromatogramma 280nm. </li><li> Pulire preconfezionati colonna in base alla produzione di raccomandazione. </li><li> SDS-PAGE analisi delle frazioni raccolte. Tale analisi deve essere eseguita con i seguenti esempi, le proteine ​​illimitata flow-through e proteine ​​picchi di deflusso. Eseguire la SDS-PAGE e macchia menzione in passi 3,17-3,18. </li><li> Valutare gel per l&#39;esistenza di proteine ​​bande al Mw previsto secondo il marcatore proteico pre-colorato. Unisci frazioni di eluizione selezionate che contengono la proteina in questione. </li><li> Dializzare i campioni fusa, in modo da abbassare le concentrazioni di NaCl necessaria per cromatografia dimensione esclusione. Eseguire la dialisi, come indicato nei passi 3,21-3,23 contro tampone F. </li><li> Concentrare il campione di proteine ​​con un Vivaspin-15 10000 MWCO a 8 mg / ml in una centrifuga da tavolo, 4000 rpm. Convalidare concentrazione desiderio da parte delle DO a 280 nm in cuvette al quarzo. Se la proteina è troppo concentrato diluirla con F tampone e misurare di nuovo. </li><li> Montare l&#39;esclusione dimensioni preconfezionati colonna, HiLoad 26/60 Superdex 200, su FPLC; lavare la colonna con 3 CV di DDW acqua filtrata. Equilibrata la colonna con 3 CV di tampone F. </li><li> Lavare ciclo di iniezione con DDW 5ml (due volumi loop) seguito da stesso volume con il tampone di F. carico non più di 4 ml del campione di proteine ​​nel ciclo di iniezione. </li><li> Iniettare il campione (portata di 3,5 ml / min) e iniziare a raccogliere frazioni di picco proteine ​​come appaiono lo cromatogramma 280nm. Lasciate che il flusso del buffer fino a raggiungere il volume della colonna. </li><li> Se il volume delle proteine ​​iniziale (passo 3.23) è più grande ansa di iniezione, ripetere i passaggi 3,25-3,26, senza lavare il ciclo, fino a quando l&#39;intero campione è stato caricato su colonna. </li><li> Pulire preconfezionati colonna secondo le raccomandazioni fabbricazione. </li><li> SDS-PAGE analisi delle frazioni raccolte. Tale analisi deve essere eseguita con i campioni di proteine ​​picchi. Eseguire la SDS-PAGE e macchia menzione in passi 3,17-3,18. </li><li> Valutare gel: se i picchi sono proteine ​​specifiche, nella corretta Mw previsto secondo il marcatore proteico pre-colorati e una sola banda è visibile unione selezionato frazioni eluizione della proteina in questione. </li><li> Concentrato campione di proteine ​​con un-15 Vivaspin 10.000 MWCO a&gt; 20 mg / ml in una centrifuga da tavolo, 4000 rpm. Convalidare concentrazione desiderio da parte delle DO a 280 nm. Il MamAΔ41 purificato è stato poi concentrato a 26,5 mg / ml per la cristallizzazione. </li> &lt;li&gt; Conferma la purezza del campione e l&#39;identificazione delle proteine ​​con Matrix-assisted laser desorbimento / ionizzazione (MALDI-TOF). </li><li> Se la proteina è purificata secondo spettrometro di massa e SDS-PAGE, dividere il MamAΔ41 concentrato di pre-marcato tubi eppendorf; 25-50μl proteina in ciascuno. </li><li> Flash congelati in azoto liquido e conservare a 193 ° K. </li></ol> 4. Rappresentante Risultati Quando questo protocollo è fatto correttamente si dovrebbe ottenere altamente purificato, campioni di proteine ​​grandezza omogenea e concentrata. Questi campioni di proteine ​​sono pronti per le prove di cristallizzazione e studi biochimici, come la cinetica enzimatica, affinità di legame e di più. Qui descritte sono risultati rappresentativi delle fasi essenziali del protocollo di purificazione. SDS-PAGE L&#39;analisi del profilo di eluizione di Ni-NTA colonna di affinità dovrebbe rivelare molto più di proteina espressa nella frazione solubile in MW appropriati di circa 22kDa (Figura 1). Questa SDS-PAGE analisi dovrebbe anche rivelare minimo eventuali proteine ​​nelle proteine ​​sconfinato flusso continuo, lavare flow-through e ultra-centrifugazione del campione di pellet. Se grandi fasce della proteina appropriati appaiono in questi campioni si dovrebbe considerare la preparazione sbagliata tampone, problemi di rottura delle cellule in coltura o problemi di auto-induzione condizioni e la crescita. Cromatografia a scambio ionico è preformato per separare tra la nostra proteina auspicabile altre proteine ​​E.coli che erano legati alla resina nichel (a causa di interazioni elettrostatiche o istidina / negativo amino acidi ricchi di proteine ​​loop) e non tagliato His-tag proteina desiderabile. Questa colonna separa le proteine ​​in base alla loro affinità di legame alla resina carica positiva con concentrazioni crescenti di NaCl. Proteina forte carica negativa si eluire in concentrazioni più elevate di NaCl appose a moderata quelli a carica negativa. I vantaggi di questa colonna sono elevata portata e capacità di legame. Il cromatogramma a scambio ionico (Figura 2) rivela una buona separazione tra i 3 popolazioni di proteine ​​per aumentare la concentrazione di NaCl. SDS-PAGE analisi è necessaria al fine di determinare MW di ciascuna popolazione, isolando quella desiderabile e valutare se le fasi di depurazione sono necessarie ulteriori. La popolazione prima è MamAΔ41 (~ 20 kDa) mentre la seconda popolazione è MamAΔ41 + Il suo Tag (~ 22kDa) che non è stata tagliata da trombina bovina e la popolazione terzo è rilevabile in SDS-PAGE a causa di bassa concentrazione. Se i picchi proteine ​​non sono separate in modo chiaro si dovrebbe considerare la preparazione sbagliata tampone o modificare la pendenza del gradiente di NaCl. Esclusione cromatografia dimensione è preformato per separare tra la nostra proteina auspicabile altre proteine ​​cellulari E.coli che erano legati al Ni-NTA resina e non sono stati separati durante la cromatografia a scambio ionico. Questa colonna separa le proteine ​​in base alle loro dimensioni. Proteine ​​di grandi dimensioni si eluire in volumi di eluizione più piccoli rispetto a quelli più piccoli che verranno eluiti in grandi volumi. Il cromatogramma colonna (figura 3) mostra una buona separazione della proteina desiderabile come una popolazione principale. La popolazione sembra eluire monomero di dimensioni adeguate con un MW di ~ 20 kDa. La presenza di banda ~ 80 kDa (proteina E.coli tipico che si lega al Ni-NTA resina) viene rilevato in SDS-PAGE prima del caricamento della colonna e scompare durante questa corsa. Poiché la concentrazione di questa proteina 80 kDa ~ è basso per cominciare, la sua popolazione non appare nel cromatogramma a causa della sua diluizione e SDS-PAGE analisi è necessaria per determinare l&#39;efficienza di purificazione. Si consiglia di caricare un campione diluito e concentrato del picco principale al SDS-PAGE così si poteva sicuramente determinare la presenza di una proteina pura in MW appropriato. A questo punto la proteina è purificata e la sua omogeneità dovrebbe essere valutata con MALDI-TOF. Questa analisi ha rivelato una proteina in MW di 20347 Da e un altro in Mw di 10.176 Da (Figura 4). I 10 kDa ~ è la proteina 20 kDa ~ raddoppiato carica. La MW previsto di MamAΔ41 con i 9 aminoacidi sinistra dopo la sua rimozione è stata-Tag Da 20596,5. Confrontandolo con il ottenute MALDI-TOF MW abbiamo scoperto che sono 248 Da parte. Questa diversità può derivare da MALDI-TOF errori di misura e / o causa di degrado comune della metionina primo e il secondo glicina. Per concludere, la proteina è altamente purificato e può essere utilizzato per ulteriori esperimenti. <img src="/files/ftp_upload/1844/1844table.jpg" alt="Tabella 1" /> Tabella 1. Formulazioni tampone. <img src="/files/ftp_upload/1844/1844fig1.jpg" alt="Figura 1" /> Figura 1. Rappresentante SDS-PAGE analisi di Ni-NTA purificazione colonna Da destra a sinistra;. P-ultra-centrifuga a pellet (3.11 passo protocollo), W-wash (3.12 passo protocollo), U-illimitata proteine ​​(3.10 passo protocollo), E- cinque corsie di eluizione samples (3,14 passo protocollo), M - marker di proteine ​​(i numeri indicano MW). La freccia indica MamAΔ41. <img src="/files/ftp_upload/1844/1844fig2.jpg" alt="Figura 2" /> Figura 2. Analisi del passo cromatografia a scambio ionico (a) a scambio ionico (colonna MonoQ) cromatogramma; Blu - 280nm assorbimento, rappresenta la concentrazione di proteine, Green-NaCl concentrazione, Rosso - l&#39;indicazione delle frazioni raccolte. (B) Rappresentante SDS-PAGE analisi della fase di purificazione a scambio ionico. Da sinistra a destra; M - marker di proteine ​​(i numeri indicano Mw), A6 - frazione primo picco, A8-secondo frazione di picco. <img src="/files/ftp_upload/1844/1844fig3.jpg" alt="Figura 3" /> Figura 3. Esclusione analisi cromatografia dimensioni (a) esclusione Dimensioni (200 Superdex colonna) cromatogramma; Blu - 280nm assorbimento, rappresenta la concentrazione di proteine, indicazione Rosso delle frazioni raccolte. (B) Rappresentante SDS-PAGE analisi di fase di purificazione esclusione dimensioni. Da sinistra a destra; M-proteina marker (i numeri indicano MW), Prei-pre-iniezione del campione, A4-6 frazione combinato raccolti dal primo picco, A4-6D-diluiti frazione raccolta dalla vetta. <img src="/files/ftp_upload/1844/1844fig4.jpg" alt="Figura 4" /> Figura 4. Matrix-assisted laser desorbimento / ionizzazione (MALDI-TOF) spettro di massa di purificato MamAΔ41. La matrice è l&#39;acido Sinapic (SA). MamAΔ41 mostrato a 20347 Da, anche mostrato è la specie raddoppiato carica di MamAΔ41 a 10176 Da.

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetosome+vesicles+are+present+before+magnetite+formation,+and+MamA+is+required+for+their+activation.">Magnetosome vesicles are present before magnetite formation, and MamA is required for their activation.</a> Proc Natl Acad Sci USA. 101, 3839-3843 (2004).</li><li>Okuda, Y., Fukumori, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+characterization+of+a+magnetosome-associated+protein,+TPR-containing+MAM22,+in+Escherichia+coli.">Expression and characterization of a magnetosome-associated protein, TPR-containing MAM22, in Escherichia coli.</a> FEBS Lett. 491, 169-173 (2001).</li><li>Taoka, A., Asada, R., Sasaki, H., Anzawa, K., Wu, L. F., Fukumori, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+localizations+of+Mam22+and+Mam12+in+the+magnetosomes+of+Magnetospirillum+magnetotacticum.">Spatial localizations of Mam22 and Mam12 in the magnetosomes of Magnetospirillum magnetotacticum.</a> J Bacteriol. 188, 3805-3812 (2006).</li><li>Studier, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+production+by+auto-induction+in+high+density+shaking+cultures.">Protein production by auto-induction in high density shaking cultures.</a> Protein Expression and Purification. 41, 207-234 (2005).</li></ol>

Purificazione di proteine ​​è il passo principale in qualsiasi biochimica delle proteine ​​o studi strutturali. Dato che ogni proteina è unico, con il proprio comportamento, si ha la necessità di definire le sue proprietà e modificare di conseguenza la sua purificazione. Obiettivo di proteine ​​deve essere analizzato come un primo passo verso la purificazione mediante strumenti bioinformatici. Sono utilizzati per calcolare la iso-elettrico punto di destinazione, valutare la sua necessità di ridurre / os...

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

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

Batteri magnetotactic comprendono un gruppo eterogeneo di microrganismi acquatici che sono in grado di orientarsi lungo i campi geomagnetici. Questo comportamento è creduto per aiutare la loro ricerca di ambienti adatti<em&gt; (1)</em&gt;. Questa capacità è conferita dal magnetosome, un organello subcellulare che consiste in una catena di montaggio lineare di vescicole lipidiche ognuno in grado di biomineralize e allegare una 50-nm ~ cristallo di magnetite o greigite. Un componente principio della magnetosome che ha dimostrato di essere necessario per la formazione di vescicole funzionale è mamma. Mama è una grande abbondanza magnetosome proteina associata, che è uno dei più caratterizzato magnetosome proteine ​​associate<em&gt; In vivo</em&gt;<em&gt; (2-6)</em&gt;. Questo articolo si concentra sulla purificazione della mamma, che nonostante in fase di studio<em&gt; In vivo</em&gt;, Nessun dettaglio chiara funzionale o strutturale sono stati identificati per questo. Bioinformatica analisi ha suggerito che la mamma è una tetra-tricopeptide ripetizione (TPR) contenente proteine. TPR è un motivo strutturale trovato come parte o formazione di una piega più grande in una vasta gamma di proteine, serve come modello per interazioni proteina-proteina e media multi-proteina complessi<em&gt; (7)</em&gt;. TPRs sono coinvolti in molte attività cruciali nei processi eucariotiche organello cellulare e molti percorsi batterica<em&gt; (8-14).</em&gt; Per capire la mamma, un TPR unico contenente proteine, proteine ​​altamente purificate è necessario, come primo passo. In questo articolo presentiamo il protocollo di purificazione di una stalla Mama cancellazione mutante (MamAΔ41) da<em&gt; M. magneticum</em&gt; AMB-1.

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

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

Mama è un unico Magnetosome proteina associata che ha dimostrato di essere coinvolti nella attivazione magnetosome. Qui vi presentiamo il protocollo di purificazione di Mama cancellazione mutante (MamAΔ41) da<em&gt; M. magneticum</em&gt; AMB-1.

Purificazione della M. magneticum Strain AMB-1 proteina Magnetosome Associated MamAΔ41

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

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

Research

JoVE Journal

Biology

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

13.4K Views.  Ben-Gurion University. Mama è un unico Magnetosome proteina associata che ha dimostrato di essere coinvolti nella attivazione magnetosome. Qui vi presentiamo il protocollo di purificazione di Mama cancellazione mutante (MamAΔ41) da M. magneticum AMB-1.

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

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

La colorazione delle proteine ​​in gel con Coomassie G-250, senza solventi organici acido acetico e

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Ricerca

Biologia

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

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

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

Utilizzando il sistema GELFREE 8100 Frazionamento per peso molecolare basata su Frazionamento con recupero della fase liquida

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

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

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

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

Espressione, solubilizzazione detergente, e purificazione di un trasportatore di membrana, la proteina multiresistente MexB Resistenza

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

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

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

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

Automated interazione idrofobica colonna di selezione cromatografia per l&#39;utilizzo in purificazione di proteine

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Purificazione di Hsp104, una proteina Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

Individuazione di partner proteine ​​interagenti mediante Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

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

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

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

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

Espressione e purificazione della proteina transmembrana cistica regolatore fibrosi conduttanza in Saccharomyces cerevisiae

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

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

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

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

Germogliamento lievito proteine ​​estrazione e purificazione per lo Studio della funzione, interazioni e modificazioni post-traduzionali

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

Identification of Post-translational Modifications of Plant Protein Complexes

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

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

Identificazione delle modificazioni post-traduzionali di proteine ​​vegetali Complessi

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

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

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

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

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