Vorremmo ringraziare la sovvenzione BE1911/8-1 della Deutsche Forschungsgemeinschaft (DFG), il progetto LOEWE GLUE e il centro di ricerca collaborativa Transport and Communication across Membranes (SFB807) per il sostegno finanziario.

<ol>
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Vengono descritti il setup e le strategie per ottimizzare l'espressione CF e l'inserimento co-traduttivo di MP funzionali nei nanodischi. L'attrezzatura necessaria comprende un bioreattore, un dispositivo di pressatura francese o simile, un lettore UV / VIS e fluorescenza, recipienti di reazione CF adatti per una configurazione a due compartimenti e un incubatore a temperatura controllata. Ulteriori attrezzature standard sono centrifughe per la raccolta di cellule di E. coli e centrifughe da tavolo che raggiungo...

Le proteine di membrana (MP) sono obiettivi impegnativi negli studi sulla produzione di proteine a causa della loro insolubilità in ambienti acquosi. Le piattaforme di produzione MP convenzionali comprendono sistemi basati su cellule come E. coli, lievito o cellule eucariotiche. Gli MP ricombinanti sintetizzati vengono estratti dalle membrane cellulari o ripiegati dai corpi di inclusione1. Dopo la solubilizzazione del detergente, gli MP possono essere trasferiti in ambienti di membrana adatti mediante protocolli di ricostituzione in vitro stabiliti. Oltre alle vescicole e ai liposomi, la ricostituzione di MP nelle membrane planari sotto forma di nanodischi2 o particelle salipro3 è diventata una tecnica di routine negli ultimi tempi. Tuttavia, tutte queste strategie implicano il contatto del detergente con i MP che può provocare destabilizzazione, dissociazione degli oligomeri e persino perdita della struttura e dell'attività proteica4. Lo screening per condizioni ottimali di solubilizzazione e ricostituzione del detergente può quindi essere noioso e richiedere molto tempo5.
La natura aperta dei sistemi cell-free (CF) consente di fornire direttamente la reazione di espressione con membrane preformate con una composizione lipidica definita. In questa modalità di espressione a base lipidica (L-CF), i MP sintetizzati hanno l'opportunità di inserire co-traduzionalmente nei doppi strati forniti 6,7 (Figura 1). I nanodischi costituiti da una proteina di scaffold di membrana (MSP) che circonda un disco planare lipidico a doppio strato8 sembrano essere particolarmente adatti per questa strategia 9,10. I nanodischi possono essere assemblati di routine in vitro con una varietà di lipidi diversi, sono molto stabili e le scorte possono essere concentrate fino a 1 mM. Tuttavia, l'espressione della MSP in E. coli e la sua purificazione sono necessarie. Come strategia alternativa, la MSP può essere co-espressa insieme al target MP nelle reazioni CF fornite con liposomi11,12,13. I modelli di DNA sia per MSP che MP vengono aggiunti nella reazione e MP/nanodischi possono formarsi durante l'espressione. Mentre la produzione di MSP è evitata, la strategia di co-espressione richiede un'attenta messa a punto delle concentrazioni finali del modello di DNA e ci si possono aspettare variazioni più elevate nell'efficienza della produzione del campione.
L'inserimento co-traduzionale di MP in membrane di nanodischi preformati è un meccanismo non fisiologico e ancora in gran parte sconosciuto indipendente dai macchinari translocon e dalle sequenze di segnale13,14,15,16. I principali determinanti dell'efficienza di inserzione sono il tipo di proteina di membrana e la composizione lipidica della membrana fornita, con una frequente preferenza per i lipidi caricati negativamente15,17. Poiché le membrane dei nanodischi sono di dimensioni relativamente limitate, una notevole quantità di lipidi viene rilasciata dopo l'inserimento MP18. La variazione della dimensione del nanodisco consente l'inserimento e la messa a punto di complessi MP oligomerici superiori15,18. Tra gli altri, è stato dimostrato il corretto assemblaggio di complessi omooligomerici per il canale ionico KcsA, per la pompa ionica proteorodopsina (PR) e per il trasportatore multifarmaco EmrE15,18. È probabile che gli MP entrino nella membrana simmetrica del nanodisco da entrambi i lati con una frequenza relativamente uguale. Va pertanto considerato che diversi monomeri o oligomeri inseriti in un nanodisco possono avere orientamenti opposti. Tuttavia, un bias nell'orientamento potrebbe essere causato da meccanismi di inserzione cooperativa come riportato per la formazione di esameri PR e tetrameri KcsA18. Un'ulteriore distorsione nell'orientamento MP potrebbe derivare da interruttori di orientamento di MP inseriti probabilmente sul bordo delle membrane del nanodisco.
La produzione di lisati CF da ceppi di E. coli è una tecnica di routine affidabile e può essere eseguita in quasi tutti i laboratori biochimici19,20. Va considerato che, oltre alla formazione di ponti disolfuro, la maggior parte delle altre modifiche post-traduzionali sono assenti se un MP viene sintetizzato usando lisati di E. coli. Sebbene ciò possa generare campioni più omogenei per gli studi strutturali, potrebbe essere necessario escludere potenziali effetti sulla funzione dei singoli bersagli MP. Tuttavia, la produzione efficiente di campioni di alta qualità di recettori accoppiati a proteine G (GPCR)14,21,22, trasportatori eucariotici 23 o grandi gruppi eteromerici 24 in lisati CF di E. coli indica la loro idoneità anche per bersagli complessi. Le quantità su scala preparativa (≈ 1 mg/ml) di un campione possono essere ottenute con la configurazione senza cella a scambio continuo a due compartimenti (CECF), composta da un compartimento di una miscela di reazione (RM) e da un compartimento di miscela di alimentazione (FM). Il volume FM supera il volume RM da 15 a 20 volte e fornisce un serbatoio di composti energetici a basso peso molecolare e precursori19. La reazione di espressione viene così estesa per diverse ore e la resa finale del target MP viene aumentata. I compartimenti RM e FM sono separati da una membrana di dialisi con un cutoff di 10-14 kDa. I due compartimenti richiedono un design speciale del contenitore di reazione CECF (Figura 1). Le cassette di dialisi commerciali come contenitori RM in combinazione con contenitori in plexiglass su misura contenenti il FM sono esempi adatti. Le rese MP possono essere ulteriormente manipolate modificando i rapporti RM:FM o scambiando l'FM dopo un certo periodo di incubazione.
La resa e la qualità di un MP richiedono spesso un'intensa ottimizzazione dei parametri di reazione. Un importante vantaggio dell'espressione CF è la possibilità di modificare e mettere a punto quasi tutti i composti in base alle esigenze individuali di un MP. Una strategia semplice consiste nel concentrarsi prima sul miglioramento della resa di un MP stabilendo un protocollo di produzione di base e quindi sull'ottimizzazione della qualità MP regolando le condizioni di reazione e piegatura. L'assenza di processi fisiologici nei lisati CF e la loro ridotta complessità si traducono in alti tassi di successo per la produzione efficiente di MP25. Le considerazioni di routine per la progettazione del modello di DNA e l'ottimizzazione della concentrazione di ioni Mg 2+ sono nella maggior parte dei casi sufficienti per ottenere rendimenti soddisfacenti26. A seconda della modalità di espressione, l'ottimizzazione della qualità MP può richiedere molto tempo, poiché è necessario esaminare una maggiore varietà di parametri14,17,22.
Per stabilire la piattaforma di espressione CF descritta, sono necessari protocolli per la produzione di E. coli CF lisato (i), T7 RNA polimerasi (ii), nanodischi (iii) e composti di reazione CECF di base (iv) (Figura 1). I derivati di E. coli K12 ceppo A1927 o BL21 sono frequentemente utilizzati per la preparazione di lisati efficienti S30 (centrifugazione a 30.000 x g). Oltre ai lisati S30, possono essere utilizzati lisati corrispondenti centrifugati ad altre forze g (ad esempio S12). I lisati differiscono nell'efficienza di espressione e nella complessità del proteoma. Il proteoma del lisato S30 preparato dal protocollo descritto è stato studiato in dettaglio28. Il proteoma S30 contiene ancora alcune proteine residue della membrana esterna che potrebbero dare problemi di fondo durante l'espressione e l'analisi dei canali ionici. Per tali obiettivi, si raccomanda l'uso di lisati S80-S100. Una preziosa modifica durante la preparazione del lisato è l'induzione della risposta SOS mediante shock termico combinato e fornitura di etanolo nella fase di crescita intermedia delle cellule. I lisati HS30 risultanti sono arricchiti in chaperoni e possono essere utilizzati in miscele con lisati S30 per migliorare il ripiegamento MP22. L'espressione di CF in lisati di E. coli è operata come un processo di trascrizione/traduzione accoppiato con modelli di DNA contenenti promotori controllati dalla T7 RNA polimerasi (T7RNAP). L'enzima può essere prodotto in modo efficiente nelle cellule stellari BL21(DE3) che trasportano il plasmide 29 pAR1219.
Due copie di MSP1E3D1 si assemblano in un nanodisco con un diametro di 10-12 nm, ma il protocollo descritto di seguito può funzionare anche per altri derivati MSP. Tuttavia, i nanodischi più grandi di quelli formati con MSP1E3D1 tendono ad essere meno stabili, mentre i nanodischi più piccoli formati con derivati MSP come MSP1 potrebbero non ospitare MP o complessi MP più grandi. I nanodischi MSP1E3D1 possono essere assemblati in vitro con una grande varietà di lipidi o miscele lipidiche diverse. I nanodischi preformati sono generalmente stabili per > 1 anno a -80 °C, mentre la stabilità può variare per diversi componenti lipidici. Per lo screening degli effetti lipidici sul ripiegamento e sulla stabilità di un MP, è necessario preparare un set completo di nanodischi assemblati con una varietà rappresentativa di miscele lipidiche / lipidiche. I seguenti lipidi possono dare una buona selezione di partenza: 1,2-Dimiristoil-sn-glicero-3-fosfo-(1'-rac-glicerolo) (DMPG), 1,2-dimiristoil-sn-glicero-3-fosfocolina (DMPC), 1,2 dioleoil-sn-glicero-3-fosfo-rac-(1-glicerolo) (DOPG), 1,2-dioleoil-sn-glicero-3-fosfocolina (DOPC), 1-palmitoil-2-oleoil-sn-glicero-3-fosfo-(1'-rac-glicerolo) (POPG) e 1-palmitoil-2-oleoil-glicero-3-fosfocolina (POPC).
Viene descritto un protocollo per la preparazione di una reazione CECF da 3 ml. È possibile un ulteriore ridimensionamento verso l'alto o verso il basso in un rapporto 1:1. Per la configurazione CECF a due compartimenti, devono essere preparati un RM contenente tutti i composti e un FM contenente solo i composti a basso peso molecolare. I dispositivi commerciali per dialisi da 3 mL con MWCO da 10-14 kDa possono essere utilizzati come contenitori RM, che vengono poi collocati in contenitori in plexiglass personalizzati contenenti FM (Figura 1D)30. Il rapporto RM:FM è 1:20, quindi 60 mL di FM devono essere preparati per 3 mL RM. La qualità, la concentrazione o il tipo di diversi componenti possono essere critici per la resa finale e/o la qualità del MP sintetizzato (Tabella 1). I modelli di DNA devono essere preparati secondo le linee guida pubblicate e l'ottimizzazione del codone del frame di lettura del bersaglio può migliorare ulteriormente significativamente la resa del prodotto26. Per la reazione CECF su scala preparativa, viene descritto un protocollo stabilito per la produzione di PR. Per stabilire protocolli di espressione per i nuovi MP, di solito è necessario eseguire schermate di ottimizzazione di alcuni composti (Tabella 1) per migliorare la resa e la qualità. Per gli ioni Mg2+ , esiste un ottimale di concentrazione ben focalizzato che spesso mostra variazioni significative per diversi modelli di DNA. Altri composti di reazione CF come nuovi lotti di lisati T7RNAP o S30 possono spostare ulteriormente la concentrazione ottimale di Mg2+ . Ad esempio, viene descritto un tipico schermo Mg 2+ nell'intervallo di concentrazione di14-24 mM e in incrementi di 2 mM. Ogni concentrazione viene sottoposta a screening in duplicati e in reazioni CECF su scala analitica. Come contenitore di reazione CECF, i contenitori in plexiglas Mini-CECF su misura30 che contengono l'RM vengono utilizzati in combinazione con micropiastre standard a 24 pozzetti che contengono l'FM (Figura 1B). In alternativa, possono essere utilizzate cartucce per dialisi commerciali in combinazione con micropiastre a pozzetti profondi 96 o altri dispositivi dializzatori commerciali in configurazioni appropriate (Figura 1C).

<table>
	<tbody>
		<tr>
			<td>1,2-dimyristoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (sodium salt) (DMPG)</td>
			<td>Avanti Polar Lipids (USA)</td>
			<td>840445P</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)</td>
			<td>Avanti Polar Lipids (USA)</td>
			<td>850345C</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-dioleoyl-sn-glycero-3-phosphocholine (sodium salt) (DOPC)</td>
			<td>Avanti Polar Lipids (USA)</td>
			<td>850375C</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2 dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (sodium salt) (DOPG)</td>
			<td>Avanti Polar Lipids (USA)</td>
			<td>840475C</td>
			<td></td>
		</tr>
		<tr>
			<td>1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC)</td>
			<td>Avanti Polar Lipids (USA)</td>
			<td>850457C</td>
			<td></td>
		</tr>
		<tr>
			<td>1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (sodium salt) (POPG)</td>
			<td>Avanti Polar Lipids (USA)</td>
			<td>840034C</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Amino-2-(hydroxymethyl)-propan-1,3-diol (Tris)</td>
			<td>Carl Roth (Germany)</td>
			<td>4855</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Carl Roth (Germany)</td>
			<td>4227</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>Carl Roth (Germany)</td>
			<td>9781</td>
			<td></td>
		</tr>
		<tr>
			<td>[3H]-dihydroalprenolol Hydrochloride</td>
			<td>American Radiolabeled Chemicals (USA)</td>
			<td>ART0134</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetyl phosphate lithium potassium salt (ACP)</td>
			<td>Merck (Germany)</td>
			<td>1409</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 5&#8217;-triphosphate (ATP)</td>
			<td>Sigma Aldrich (Germany)</td>
			<td>A9251</td>
			<td></td>
		</tr>
		<tr>
			<td>Alprenolol hydrochloride</td>
			<td>Merck (Germany)</td>
			<td>A0360000</td>
			<td></td>
		</tr>
		<tr>
			<td>Anion exchange chromatography column material: Q-sepharose&#174;</td>
			<td>Sigma-Aldrich (Germany)</td>
			<td>Q1126</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave Type GE 446EC-1</td>
			<td>Gettinge (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bioreactor Type 884 124/1</td>
			<td>B.Braun (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Sorvall RC12BP+; Thermo Scientific (Germany); Sorvall RC-5C; Thermo Scientific (Germany); Mikro 22 R; Hettich (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cholic acid</td>
			<td>Carl Roth (Germany)</td>
			<td>8137</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue R250</td>
			<td>Carl Roth (Germany)</td>
			<td>3862</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture flasks 500 ml baffled flasks, 2 l baffled flasks</td>
			<td>Schott Duran (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cytidine 5&#39;-triphosphate disodium salt</td>
			<td>Sigma-Aldrich (Germany)</td>
			<td>C1506</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose monohydrate</td>
			<td>Carl Roth (Germany)</td>
			<td>6780</td>
			<td></td>
		</tr>
		<tr>
			<td>Di-potassiumhydrogen phosphate trihydrate</td>
			<td>Carl Roth (Germany)</td>
			<td>6878</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis tubing SpectrumTM Labs Spectra/PorTM 12-14 kD MWCO Standard RC tubing</td>
			<td>Fisher Scientific (Germany)</td>
			<td>8700152</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreit</td>
			<td>Carl Roth (Germany)</td>
			<td>6908</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Carl Roth (Germany)</td>
			<td>K928</td>
			<td></td>
		</tr>
		<tr>
			<td>Folinic acid calcium salt hydrate</td>
			<td>Sigma-Aldrich (Germany)</td>
			<td>47612</td>
			<td></td>
		</tr>
		<tr>
			<td>French pressure cell disruptor</td>
			<td>SLM; Amico Instruments (USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Carl Roth (Germany)</td>
			<td>3783</td>
			<td></td>
		</tr>
		<tr>
			<td>Guanosine 5&#39;-triphosphate di-sodium salt (GTP)</td>
			<td>Sigma-Aldrich (Germany)</td>
			<td>G8877</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Carl Roth (Germany)</td>
			<td>K025</td>
			<td></td>
		</tr>
		<tr>
			<td>IMAC column: HiTrap IMAC HP 5 mL</td>
			<td>GE Life Sciences (Germany)</td>
			<td>GE17-5248</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Carl Roth (Germany)</td>
			<td>3899</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl-&#946;-D-thiogalactopyranosid (IPTG)</td>
			<td>Carl Roth (Germany)</td>
			<td>2316</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Carl Roth (Germany)</td>
			<td>T832</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Alanine</td>
			<td>Carl Roth (Germany)</td>
			<td>3076.1</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Arginine</td>
			<td>Carl Roth (Germany)</td>
			<td>6908</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Asparagine</td>
			<td>Carl Roth (Germany)</td>
			<td>HN23</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Aspartic Acid</td>
			<td>Carl Roth (Germany)</td>
			<td>T202</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Carl Roth (Germany)</td>
			<td>T203</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamic Acid</td>
			<td>Carl Roth (Germany)</td>
			<td>3774</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Carl Roth (Germany)</td>
			<td>3772</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glycine</td>
			<td>Carl Roth (Germany)</td>
			<td>3187</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Histidine</td>
			<td>Carl Roth (Germany)</td>
			<td>3852</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Isoleucine</td>
			<td>Carl Roth (Germany)</td>
			<td>3922</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Leucine</td>
			<td>Carl Roth (Germany)</td>
			<td>1699</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Lysine</td>
			<td>Carl Roth (Germany)</td>
			<td>4207</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Methionine</td>
			<td>Carl Roth (Germany)</td>
			<td>9359</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Proline</td>
			<td>Carl Roth (Germany)</td>
			<td>1713</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Phenylalanine</td>
			<td>Carl Roth (Germany)</td>
			<td>1709</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Serine</td>
			<td>Carl Roth (Germany)</td>
			<td>4682</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Threonine</td>
			<td>Carl Roth (Germany)</td>
			<td>1738</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Tryptophane</td>
			<td>Carl Roth (Germany)</td>
			<td>7700</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Tyrosine</td>
			<td>Carl Roth (Germany)</td>
			<td>T207</td>
			<td></td>
		</tr>
		<tr>
			<td>MD100 dialysis units</td>
			<td>Scienova (Germany)</td>
			<td>40077</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2-Hydroxyethylpiperazine-N&#39;-2-ethansulfonic acid (HEPES)</td>
			<td>Carl Roth (Germany)</td>
			<td>6763</td>
			<td></td>
		</tr>
		<tr>
			<td>n-dodecylphosphocholine</td>
			<td>Antrace (USA)</td>
			<td>F308S</td>
			<td></td>
		</tr>
		<tr>
			<td>PAGE chamber: Mini-Protean Tetra Cell</td>
			<td>Biorad (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PAGE gel casting system: Mini Protean Handcast systems</td>
			<td>Biorad (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PAGE gel power supply: Power Pac 3000</td>
			<td>Biorad (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone/peptone from caseine</td>
			<td>Carl Roth (Germany)</td>
			<td>6681</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump: ip-12</td>
			<td>Ismatec (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphoenol pyruvate monopotassium salt</td>
			<td>Sigma Aldrich (Germany)</td>
			<td>860077</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate</td>
			<td>Carl Roth (Germany)</td>
			<td>P018</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium acetate</td>
			<td>Carl Roth (Germany)</td>
			<td>4986</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Carl Roth (Germany)</td>
			<td>6781</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyruvate Kinase</td>
			<td>Roche (Germany)</td>
			<td>10109045001</td>
			<td></td>
		</tr>
		<tr>
			<td>Scintillation counter: Hidex 300 SL</td>
			<td>Hidex (Finland)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SDS pellets</td>
			<td>Carl Roth (Germany)</td>
			<td>8029</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich (Germany)</td>
			<td>K305</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Carl Roth (Germany)</td>
			<td>P029</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer Nanodrop</td>
			<td>Peqlab (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer/fluorescence reader Spark&#174;</td>
			<td>Tecan (Switzerland)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>tRNA (E. coli)</td>
			<td>Roche (Germany)</td>
			<td>10109550001</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra sonificator</td>
			<td>Labsonic U, B. Braun (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine 5&#8217;-triphosphate tri-sodium salt (UTP)</td>
			<td>Sigma-Aldrich (Germany)</td>
			<td>U6625</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-30 antifoam</td>
			<td>Sigma-Aldrich (Germany)</td>
			<td>A6457</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Carl Roth (Germany)</td>
			<td>2904</td>
			<td></td>
		</tr>
	</tbody>
</table>

co-translational insertion of membrane proteins into preformed nanodiscs

I sistemi di espressione cell-free consentono la progettazione su misura di ambienti di reazione per supportare il ripiegamento funzionale di proteine anche complesse come le proteine di membrana. Vengono dimostrate le procedure sperimentali per l'inserimento e il ripiegamento co-traduzionale di proteine di membrana in membrane preformate e definite fornite come nanodischi. Il protocollo è completamente privo di detergenti e può generare milligrammi di campioni purificati in un giorno. I campioni di proteine di membrana/nanodisco risultanti possono essere utilizzati per una varietà di studi funzionali e applicazioni strutturali come la cristallizzazione, la risonanza magnetica nucleare o la microscopia elettronica. Viene descritta la preparazione di componenti chiave di base come lisati cell-free, nanodischi con membrane progettate, soluzioni stock critiche e l'assemblaggio di reazioni di espressione prive di cellule a due compartimenti. Poiché i requisiti di ripiegamento delle proteine di membrana possono essere molto diversi, un obiettivo principale di questo protocollo è la modulazione dei parametri e delle fasi di reazione importanti per la qualità del campione come i composti critici di reazione di base, la composizione della membrana dei nanodischi, l'ambiente redox e chaperone o la progettazione del modello di DNA. L'intero processo è dimostrato con la sintesi di proteorodopsina e un recettore accoppiato alla proteina G.

L'impatto dei composti di reazione di messa a punto sulla resa finale o sulla qualità dei MP sintetizzati è esemplificato. Un approccio di routine frequente è quello di regolare la concentrazione ottimale di Mg2+ nelle reazioni CF mediante l'espressione della proteina fluorescente verde (GFP) come un comodo monitor dell'efficienza del sistema. Ad esempio, la GFP è stata sintetizzata da un vettore pET-21a (+) a concentrazioni di Mg 2+ comprese tra 14 e24 mM (Figura 2...

Watch this Scientific Journal Video about Co-Translational Insertion of Membrane Proteins into Preformed Nanodiscs at JoVE.com

Co-Translational Insertion of Membrane Proteins into Preformed Nanodiscs

L'inserimento co-traduzionale in nanodischi preformati rende possibile lo studio di proteine di membrana sintetizzate prive di cellule in ambienti lipidici definiti senza contatto con detergenti. Questo protocollo descrive la preparazione dei componenti essenziali del sistema e i parametri critici per migliorare l'efficienza dell'espressione e la qualità del campione.

Inserimento co-traduzionale di proteine di membrana in nanodischi preformati

Cell-free expression systems allow the tailored design of reaction environments to support the functional folding of even complex proteins such as ...

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3.2K Views.  Goethe University Frankfurt. L'inserimento co-traduzionale in nanodischi preformati rende possibile lo studio di proteine di membrana sintetizzate prive di cellule in ambienti lipidici definiti senza contatto con detergenti. Questo protocollo descrive la preparazione dei componenti essenziali del sistema e i parametri critici per migliorare l'efficienza dell'espressione e la qualità del campione.

Video: Co-Translational Insertion of Membrane Proteins into Preformed Nanodiscs

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Utilizzando impalcatura liposomi per ricostituire interazioni proteina-proteina lipidi-prossimale<em&gt; In Vitro</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

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Biochimica

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

Metodo per visualizzare e analizzare membrana proteine ​​interagenti con microscopia elettronica a trasmissione

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

Nitrogen Cavitation and Differential Centrifugation Allows for Monitoring the Distribution of Peripheral Membrane Proteins in Cultured Cells

Cultured cells are useful for studying the subcellular distribution of proteins, including peripheral membrane proteins. Genetically encoded fluorescently tagged proteins have revolutionized the study of subcellular protein distribution. However, it is difficult to quantify the distribution with fluorescent microscopy, especially when proteins are partially cytosolic. Moreover, it is often important to study endogenous proteins. Biochemical assays such as immunoblots remain the gold standard for quantification of protein distribution after subcellular fractionation. Although there are commercial kits that aim to isolate cytosolic or certain membrane fractions, most of these kits are based on extraction with detergents, which may be unsuitable for studying peripheral membrane proteins that are easily extracted from membranes. Here we present a detergent-free protocol for cellular homogenization by nitrogen cavitation and subsequent separation of cytosolic and membrane-bound proteins by ultracentrifugation. We confirm the separation of subcellular organelles in soluble and pellet fractions across different cell types, and compare protein extraction among several common non-detergent-based mechanical homogenization methods. Among several advantages of nitrogen cavitation is the superior efficiency of cellular disruption with minimal physical and chemical damage to delicate organelles. Combined with ultracentrifugation, nitrogen cavitation is an excellent method to examine the shift of peripheral membrane proteins between cytosolic and membrane fractions.

Here we present protocols for detergent-free homogenization of cultured mammalian cells based on nitrogen cavitation and subsequent separation of cytosolic and membrane-bound proteins by ultracentrifugation. This method is ideal for monitoring the partitioning of peripheral membrane proteins between soluble and membrane fractions.

Cavitazione di azoto e centrifugazione differenziale permette di monitorare la distribuzione periferica delle proteine di membrana in cellule coltivate

Cultured cells are useful for studying the subcellular distribution of proteins, including peripheral membrane proteins. Genetically encoded fluorescently ...

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers are less compatible with detergents.
Here we describe a strategy for bypassing this fundamental limitation using fusogenic oppositely charged liposomes bearing a membrane protein of interest. Fusion between such vesicles occurs within 5 min in a low ionic strength buffer. Positively charged fusogenic liposomes can be used as simple shuttle vectors for detergent-free delivery of membrane proteins into biomimetic target lipid bilayers, which are negatively charged. We also show how to reconstitute membrane proteins into fusogenic proteoliposomes with a fast 30-min protocol.
Combining these two approaches, we demonstrate a fast assembly of an electron transport chain consisting of two membrane proteins from E. coli, a primary proton pump bo3-oxidase and F1Fo ATP synthase, in membranes of vesicles of various sizes, ranging from 0.1 to &#62;10 microns, as well as ATP production by this chain.

Here we present two ultrafast protocols for reconstitution of membrane proteins into fusogenic proteoliposomes, and fusion of such proteoliposomes with target lipid bilayers for detergent-free delivery of these membrane proteins into the postfusion bilayer. The combination of these approaches enables fast and easily controlled assembly of complex multi-component membrane systems.

Privo di detersivo ultraveloce ricostituzione delle proteine di membrana in lipidici utilizzando fusogena proteoliposomi complementari a carico.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers ...

Detection of Detergent-sensitive Interactions Between Membrane Proteins

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein interactions in many cases is associated with significant experimental challenges. In particular, sorting receptors interact with their protein cargo in the lumen of the membrane compartments often in a detergent-sensitive fashion, making co-immunoprecipitation of these proteins unusable. Binding of the sorting receptor sortilin to glucose transporter GLUT4 may serve as an example of weak luminal interactions between membrane proteins. Here, we describe a fast, simple, and inexpensive assay to validate the interaction between sortilin and GLUT4. For that, we have designed and chemically synthesized the myc-tagged peptide corresponding to the potential sortilin-binding epitope in the luminal part of GLUT4. Sortilin tagged with six histidines was expressed in mammalian cells, and isolated from cell lysates using Cobalt beads. Sortilin immobilized on the beads was incubated with the peptide solution at different pH values, and the eluted material was analyzed by Western blotting. This assay can be easily adapted to study other detergent-sensitive protein-protein interactions.

We describe a protocol for detection of detergent-sensitive interactions between membrane proteins using binding of the sorting receptor, sortilin, to the first luminal loop of the glucose transporter protein, GLUT4, as an example.

Rilevamento di detersivo sensibili interazioni tra proteine di membrana

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

Un'analisi di spettroscopia di fluorescenza fluttuazione delle interazioni proteina-proteina a contatti cellula-cellula

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

PeptiQuick, a One-Step Incorporation of Membrane Proteins into Biotinylated Peptidiscs for Streamlined Protein Binding Assays

Membrane proteins, including transporters, channels, and receptors, constitute nearly one-fourth of the cellular proteome and over half of current drug targets. Yet, a major barrier to their characterization and exploitation in academic or industrial settings is that most biochemical, biophysical, and drug screening strategies require these proteins to be in a water-soluble state. Our laboratory recently developed the peptidisc, a membrane mimetic offering a &#8220;one-size-fits-all&#8221; approach to the problem of membrane protein solubility. We present here a streamlined protocol that combines protein purification and peptidisc reconstitution in a single chromatographic step. This workflow, termed PeptiQuick, allows for bypassing dialysis and incubation with polystyrene beads, thereby greatly reducing exposure to detergent, protein denaturation, and sample loss. When PeptiQuick is performed with biotinylated scaffolds, the preparation can be directly attached to streptavidin-coated surfaces. There is no need to biotinylate or modify the membrane protein target. PeptiQuick is showcased here with the membrane receptor FhuA and antimicrobial ligand colicin M, using biolayer interferometry to determine the precise kinetics of their interaction. It is concluded that PeptiQuick is a convenient way to prepare and analyze membrane protein-ligand interactions within one&#160;day in a detergent-free environment.

We present a method that combines membrane protein purification and reconstitution into peptidiscs in a single chromatographic step. Biotinylated scaffolds are used for direct surface attachment and measurement of protein-ligand interactions via biolayer interferometry.

PeptiQuick, un'incorporazione in un solo passaggio di proteine membrana in Peptidiscbio biotinylated per analisi di legame proteico semplificato

Membrane proteins, including transporters, channels, and receptors, constitute nearly one-fourth of the cellular proteome and over half of current drug ...

Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular mechanism for learning and memory. Palmitoylation regulates localization and function of many synaptic proteins including AMPA-Rs, auxiliary factors and synaptic scaffolds in an activity-dependent manner. We identified the synapse differentiation induced gene (SynDIG) family of four genes (SynDIG1-4) encoding brain-specific transmembrane proteins that associate with AMPARs and regulate synapse strength. SynDIG1 is palmitoylated at two cysteine residues located at positions 191 and 192 in the juxta-transmembrane region important for activity-dependent excitatory synapse development. Here, we describe an innovative biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest and demonstrate its utility with the SynDIG family of proteins in mouse brain lysates.

Palmitoylation entails the incorporation of a 16-carbon palmitate moiety to cysteine residues of target proteins in a reversible manner. Here, we describe a biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest in mouse brain lysates.

Acyl-PEGyl Exchange Gel Shift Analisi per la Determinazione Quantitativa della Palmitoylation delle Proteine della Membrana Cerebrale

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular ...

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. This method combines expression of proteins tagged with the fluorescent non-canonical amino acid ANAP, and FRET between ANAP and fluorescent (trinitrophenyl) nucleotide derivatives. We present examples of nucleotide binding to ANAP-tagged KATP ion channels measured in unroofed plasma membranes and excised, inside-out membrane patches under voltage clamp. The latter allows for simultaneous measurements of ligand binding and channel current, a direct readout of protein function. Data treatment and analysis are discussed extensively, along with potential pitfalls and artefacts. This method provides rich mechanistic insights into the ligand-dependent gating of KATP channels and can readily be adapted to the study of other nucleotide-regulated proteins or any receptor for which a suitable fluorescent ligand can be identified.

This protocol presents a method for measuring adenine nucleotide binding to receptors in real time in a cellular environment. Binding is measured as F&#246;rster resonance energy transfer (FRET) between trinitrophenyl nucleotide derivatives and protein labeled with a non-canonical, fluorescent amino acid.

Misurare il legame nucleotidico a proteine di membrana intatte e funzionali in tempo reale

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm phenotyping can be used to classify genotypes based on SG morphotype using scanning electron microscopic (SEM) analysis. SGs can be visualized using SEM by slicing through the kernel (pericarp, aleurone layers, and endosperm) and exposing the organellar contents. Current methods require the rice kernel to be embedded in plastic resin and sectioned using a microtome or embedded in a truncated pipette tip and sectioned by hand using a razor blade. The former method requires specialized equipment and is time-consuming, while the latter introduces a new host of problems depending on rice genotype. Chalky rice varieties, particularly, pose a problem for this type of sectioning due to the friable nature of their endosperm tissue. Presented here is a technique for preparing translucent and chalky rice kernel sections for microscopy, requiring only pipette tips and a scalpel blade. Preparing the sections within the confines of a pipette tip prevents rice kernel endosperm from shattering (for translucent or &#8216;vitreous&#8217; phenotypes) and crumbling (for chalky phenotypes). Using this technique, endosperm cell patterning and the structure of intact SGs can be observed.

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.

Sezionamento trasversale di riso maturo (Oryza sativa L.) Kernel per la scansione di immagini al microscopio elettronico utilizzando le punte delle pipette come supporto per l'immobilizzazione

Methods Article

Inserimento co-traduzionale di proteine di membrana in nanodischi preformati