Questo progetto è stato finanziato da sovvenzioni NIH EY025290, GM127652 e Università di Rochester finanziamento di Start-up.

1. produzione BacMam espressione Baculoviridae
<ol><li>Inserire la sequenza di codificazione di una proteina desiderata bestrophin mammiferi il piolo BacMam vector18 con una sequenza di riconoscimento della proteasi Tobacco Etch Virus (TEV), seguita da un GFP-10 x His-tag al C-terminale della proteina.</li>
	<li>Transitoriamente transfect il plasmide di espressione in adesivo HEK293 cellule19,20,21,22,23. Verificare che l'espressione della proteina GFP-fusione sotto un microscopio a fluorescenza con 10 X o 20 ingrandimenti, una sorgente di eccitazione 488 nm e un filtro di emissione 510 nm (Figura 1A). Nota: A base di polimeri transfezione è effettuata ordinariamente con 1 μg di DNA del plasmide per un piatto di 35 mm di cellule HEK293.</li>
	<li>Produrre i Baculoviridae in cellule di insetto Sf9 come precedentemente descritto16,17. Nota: Il passaggio 3 (P3) virus, che verrà utilizzato per infettare le cellule HEK293-F per la produzione di proteine, può essere memorizzato a 4 ° C per 1 mese.</li>
	<li>Determinare il titolo (unità infettive) del virus P3 la placca formazione dosaggio16,17. Nota: Come la proteina di interesse è etichettata con GFP, un metodo più veloce per il controllo di unità infettive è di infettare le cellule Sf9 con diluizioni seriali del virus e contare il numero di cellule fluorescenti dopo 48 h.</li>
</ol>2. espressione della proteina
<ol><li>Mantenere le cellule HEK293-F in una cultura di sospensione con il mezzo di espressione HEK293-F in un incubatore a 37 ° C con 8% CO2. Utilizzare matracci di cultura usa e getta che sono 2 - 2,5 volte più grande rispetto al volume di cultura e ruotare la cultura su una piattaforma orbitale a 135 giri/min. Nota: Si consiglia di infettare le cellule quando essi sono tra i passaggi 5 e 30 e per eseguire queste azioni sotto una cappa di cultura cellulare.</li>
	<li>24 h prima dell'infezione virale, aggiungere 15 μL di Trypan blu ad un'aliquota di 15 μL di coltura cellulare e controllare la densità delle cellule e l'attuabilità utilizzando un emocitometro sotto un microscopio. Diluire le cellule a 0,6 x 106 cellule/mL in beute di 2 x 2 L ciascuna, con 500 mL di terreno pre-riscaldato a 37 ° C. Nota: Le cellule morte sono macchiate blu di Trypan blue. L'attuabilità delle cellule desiderata è > 95%.</li>
	<li>Il giorno di infezione, controllare la densità delle cellule e l'attuabilità (punto 2.2). Nota: Un alta attuabilità delle cellule di > 95% è essenziale per l'efficace espressione della proteina e di infezione. La densità di cella prevista è ~1.0 x 106 cellule/mL (adulto pernottamento da 2 x 500 mL 0,6 x coltura cellulare di 106 cellule/mL). Il numero totale previsto di celle è ~1.0 x 109.</li>
	<li>Aggiungere i Baculoviridae P3 le cellule ad una molteplicità di infezione (MOI) di 5. Per determinare la quantità di virus per inoculare, utilizzare la seguente equazione: <img alt="Equation" src="/files/ftp_upload/57832/57832eq1.jpg">
</li>
	<li>Agitare le cellule sulla piattaforma orbitale a 135 giri/min in un incubatore umidificato 37 ° C con 8% CO2 per 24 h.</li>
	<li>Aggiungere 10 mM del butirrato del sodio alla coltura delle cellule e continuare in incubazione per 48 h. Nota: Per alcune proteine, riducendo la temperatura di coltura cellulare a 30 ° C dopo l'aggiunta di sodio butirrato può migliorare espressione e stabilità.</li>
	<li>Microscopio a fluorescenza con 10x o 20x ingrandimento, una sorgente di eccitazione 488 nm e un filtro di emissione 510 nm, controllare la percentuale e la luminosità delle cellule verdi, che rappresentano direttamente la resa di proteina (bestrophin-GFP-10xHis) (Figura 1B). Nota: La percentuale di cellule verde viene calcolata da: 100 x (cellule cellule verde/totale). Livello di espressione di proteine buona è indicato forte fluorescenza verde sotto il microscopio.</li>
	<li>Raccogliere le cellule mediante centrifugazione a 1.000 x g a 4 ° C per 20 minuti rimuovere il surnatante e risospendere il pellet cellulare con tampone fosfato salino (PBS) ad un volume finale di ~ 80 mL. Split la sospensione cellulare in provette coniche da 2 x 50 mL e centrifugare a 1.000 x g a 4 ° C per 20 min. memorizzare il pellet di cellule a-80 ° C.</li>
</ol>3. protein Purification
<ol><li>Incubare le provette coniche contenente il pellet cellulare in un bagnomaria agitazione a temperatura ambiente per 10-15 min, in modo che si sciolga. Risospendere le cellule in volume (p/v), del buffer A (tabella 1) (ad es., 10 g di palline delle cellule in 20 mL di tampone), completati con inibitori della proteasi (fluoruro di aprotinina, leupeptina, Pepstatin A e phenylmethylsulfonyl): 2x a 0.1-1.0 mM. Dispensare su e giù estesamente per ottenere una sospensione cellulare omogenea.</li>
	<li>Lisare le cellule utilizzando un omogeneizzatore ad alta pressione. Eseguire la sospensione cellulare attraverso l'omogeneizzatore a 7-10 MPa per un totale di 3 - 4 volte per ottenere la completa omogeneizzazione. Mantenere la cella lysate sul ghiaccio per 2-3 minuti tra un round.</li>
	<li>Aggiungere detersivo [ad es., 2% w/v sol-grado n-dodecil-β-D-Maltopyranoside (DDM)] il lysate delle cellule. Incubare con agitazione per 1 h a 20 ° C a estrarre le proteine di membrana. Nota: Il tipo di detergente deve essere ottimizzato per ogni proteina bersaglio18,24.</li>
	<li>Centrifugare a 150.000 x g in un'ultracentrifuga a 4 ° C per 1 h. Nota: Tutte le seguenti operazioni vengono eseguite a 4 ° C.</li>
	<li>Raccogliere il lysate delle cellule chiare e il flusso attraverso una colonna di affinità 5ml - NTA2 +alla sua trappola Ni equilibrato con buffer A (tabella 1). Nota: Dopo la centrifugazione, il lysate delle cellule chiaro verrà essere inserita tra una pallina nella parte inferiore e uno strato nuvoloso sulla parte superiore. Utilizzare una pipetta di trasferimento di 10 mL per raccogliere con cura il chiaro lisato e quindi passare a una pipetta da 1 mL per l'ultimo pochi millilitri di lisato. Evitare il pellet, che possa ostruire la colonna2 + Ni; Tuttavia, una piccola quantità di strato superiore va bene o può essere filtrata con un filtro di 1,5 μm.</li>
	<li>Lavare la colonna con 25 mL di tampone B e poi 40 mL di tampone C (tabelle 2 e 3, rispettivamente). Nota: Questo è un buon posto per prendere una pausa, come purificazione completa può richiedere fino a 12 h e la proteina può rimanere stabile mentre è collegato alla colonna durante la notte.</li>
	<li>Fissare la colonna a un sistema di cromatografia in fase liquida (FPLC) proteine veloci ed eluire la proteina dalla colonna con 13 mL di tampone D (tabella 3) con il frazionamento. Raccogliere le frazioni proteici secondo la lettura di assorbanza UV. Nota: The FPLC condizioni sono le seguenti: un formato di frazione 1,0 mL, un allarme di pressione pre-colonna impostato su 0,3 MPa e una portata di 0,5 mL/min.</li>
	<li>Misurare la concentrazione di prodotto proteine eluite su uno spettrofotometro di microvolume. Leggere l'assorbanza a 280 nm.</li>
	<li>(Opzionale) Per rimuovere il tag di GFP-10xHis, aggiungere la proteasi TEV in un rapporto di 1:1 massa e incubare a 4 ° C per 30 min. Nota: Il tag può o non può influenzare la funzione o la struttura del canale purificato. Calcolare la quantità TEV dal volume e la concentrazione della proteina raccolta da passaggi 3.7-3.8. Per esempio, se 10 mL di prodotto di eluizione è raccolto e misurato a 0,1 mg/mL, la proteina totale massa è 10 x 0,1 = 1 mg e 1 mg di TEV verrà utilizzato per la digestione.</li>
	<li>Concentrare la proteina con un 15 mL centrifuga (50 o 100 kDa) unità filtro di filatura a 4.000 x g a 4 ° C fino ad un volume finale di 400-500 μL (per un ciclo di caricamento del campione 2 mL su FPLC). Nota: Il tempo di centrifugazione per concentrazione varia a seconda della concentrazione e il formato della proteina. Per evitare l'eccessiva concentrazione, che può causare precipitazione della proteina, gira per 10 min in un primo momento, poi osservare il volume residuo e stimare il tempo per un giro successivo (ad es., 2-5 min), e così via, per ottenere il volume finale. Pipettare tra spin per disperdere la proteina, che è tirata verso il basso del filtro.</li>
	<li>Trasferire la proteina concentrata per un nuovo 1,5 mL tubo e rimuovere qualsiasi precipitato o bolle dal prodotto concentrato. Rotazione a > 12.000 x g per 5-10 min a 4 ° C e raccogliere il surnatante in una nuova provetta da 1,5 mL.</li>
	<li>Caricare il prodotto finale con una siringa da 1 mL e un ago di turno-punta su un sistema FPLC per la cromatografia di esclusione dimensionale con una colonna di esclusione dimensionale equilibrata con buffer E (tabella 5). Nota: The FPLC condizioni sono le seguenti: un formato di frazione di 0,5 mL, un allarme di pressione pre-colonna impostata su 2,50 MPa, un volume di flusso impostato a 30 mL di tampone E (tabella 5), e una portata impostata su 0,5 mL/min.</li>
	<li>Si noti che proteine ben educati eseguire come picco singolo (Figura 2A) e raccolgono il fraction(s) della proteina corrispondente a quel picco (Figura 2A).</li>
	<li>Concentrare la proteina con un'unità di filtro centrifugo da 4 mL o 0,5 mL (della linea di demarcazione peso molecolare stesso come descritto al punto 3.10) e spin a 4.000 x g a 4 ° C per una concentrazione finale di 5-10 μg/μL. Utilizzare lo spettrofotometro per controllare la concentrazione del prodotto finale (punto 3.8). Nota: Tempo di centrifugazione varia, come il volume del prodotto finale può differire tra le prove di purificazione. Solitamente, centrifugazione prende 10-20 min. Si raccomanda di dispensare tra spin per disperdere la proteina, che è tirata verso il basso del filtro.</li>
	<li>Trasferire le proteine concentrate in una nuova provetta da 1,5 mL. Rimuovere qualsiasi precipitato mediante centrifugazione a > 12.000 x g per 5-10 min a 4 ° C. Trasferire il surnatante in una nuova provetta da 1,5 mL. 10 aliquote del μL per la conservazione a-80 ° C e risparmia un 2-5 μL piccola aliquota per eseguire su un gel di SDS-PAGE 4-15% pendenza a 9 V/cm (Figura 2B).</li>
	<li>Confermare l'identità della proteina purificata di spettrometria di massa8. Nota: Analisi In vitro della funzione e della struttura della proteina purificata può essere eseguite attraverso il doppio strato lipidico planare e cristallografia a raggi x, rispettivamente5,8.</li>
</ol>

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Questo protocollo descrive una pipeline utile per espressione e purificazione dei canali ionici bestrophin mammiferi da utilizzare per le analisi future in vitro . Mentre il dispositivo FPLC è richiesto per la cromatografia di esclusione dimensionale, una pompa a siringa è sufficiente per tutti i passaggi di cromatografia di affinità tra cui associazione, lavaggio e medicati. Quando si utilizza una pompa a siringa a soluzioni di push (in una siringa) attraverso una colonna, è essenziale per sottofondo il lat...

Bestrophins sono una famiglia di canali ionici conservata attraverso specie variano da batteri a esseri umani1. In esseri umani, del gene BEST1 , localizzato sul cromosoma 11q12.3, codifica per la proteina di membrana Bestrophin-1 (BEST1) che si esprime principalmente nella membrana basolaterale delle cellule epitelio retinico del pigmento del occhi2,3 ,4. Composto da 585 aminoacidi, il primo ~ 350 di cui sono altamente conservati tra specie e contenere la sua regione transmembrana, BEST1 agisce come un CaCC in esseri umani1,5,6. Inoltre, gli omologhi BEST1 nei polli e Klebsiella pneumoniae entrambi funzionano come homopentamers7,8, suggerendo un alto livello di conservazione nel corso dell'evoluzione.
In esseri umani, oltre 200 mutazioni del gene BEST1 sono stati clinicamente collegate ad un gruppo di malattie di degenerazione retinica denominato bestrophinopathies1,9. Cinque bestrophinopathies specifici sono stati segnalati, compreso la malattia di Best, distrofia vitelliforme di adulto-inizio, Vitreoretinocoroidopatia autosomica dominante, autosomica recessiva Bestrofinopatia e retinite pigmentosa3,4 ,10,11,12,13,14. Queste malattie, che conducono ad una riduzione della vista e persino cecità, sono attualmente incurabili. Al fine di sviluppare trattamenti terapeutici e medicina potenzialmente personalizzata, è fondamentale per capire come queste mutazioni malattia-causanti BEST1 influenzano la funzione e la struttura del BEST1 canale15. Per questi scopi, i ricercatori devono ottenere canali purificata bestrophin (wild-type e/o mutante) e condotta in vitro analisi5,8.
Il primo passo fondamentale è l'espressione di canali bestrophin da specie superiori in cellule di mammifero. Come baculovirus trasduzione di cellule HEK293-F (sistema BacMam) è un metodo potente per esprimere heterologously membrana proteine16,17, questo protocollo utilizza un vettore di BacMam ottimizzato (pEG BacMam) per robusta espressione della destinazione della proteina18, che in questo caso è un omologo mammifero bestrophin. Questo vettore è stato utilizzato per l'espressione di varie proteine di membrana, tra cui i recettori accoppiati a proteine G, recettori nucleari e altri canali ion18. C'è inoltre la prova che le proteine prodotte sono adatte per cristallografia18. Con alti livelli di espressione in cellule HEK293-F, le proteine possono essere purificate poi mediante cromatografia; in particolare, nel caso di bestrophins, affinità e la cromatografia di esclusione dimensionale può essere utilizzati.
Una volta che questo protocollo è messa a punto per un canale di bestrophin, la proteina purificata possa poi essere analizzata per la sua funzione e la struttura attraverso il doppio strato lipidico planare e cristallografia a raggi x, rispettivamente5,8. Complessivamente, queste tecniche forniscono una potente pipeline per indagini strutturali e funzionali di bestrophins e altri canali ionici.

<table border="0" cellpadding="0" cellspacing="0" width="680">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES</td>
			<td style="width:148px;">Fisher Scientific</td>
			<td style="width:119px;">AC327265000&#160;</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">AC446212500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycerol</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">G33-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Imidazole</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">AC301870010</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">MgCl2</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">AC197530010&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TCEP</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">AA4058704&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Aprotinin</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">AAJ63039MA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leupeptin</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">AAJ61188MB&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pepstatin A</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">AAJ20037MB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phenylmethylsulfonyl fluoride</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">AC215740050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DDM sol-grade</td>
			<td style="width:148px;">Anatrace</td>
			<td style="width:119px;">D310S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DDM anagrade</td>
			<td style="width:148px;">Anatrace</td>
			<td style="width:119px;">D310</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sf-900 II SFM</td>
			<td style="width:148px;">ThermoFisher</td>
			<td style="width:119px;">10902179</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FreeStyle medium</td>
			<td>ThermoFisher</td>
			<td style="width:119px;">12338018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoDrop spectrophotometer</td>
			<td>ThermoFisher</td>
			<td style="width:119px;">ND-2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">High pressure homogenizer</td>
			<td style="width:148px;">Avestin</td>
			<td style="width:119px;">Emulsiflex-C5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HisTrap column</td>
			<td style="width:148px;">GE</td>
			<td>17-5248-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superdex-200 column</td>
			<td style="width:148px;">GE</td>
			<td style="width:119px;">28990944</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AKTA Pure</td>
			<td style="width:148px;">GE</td>
			<td style="width:119px;">29018224</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultra-15 centrifugal filter units</td>
			<td style="width:148px;">Millipore</td>
			<td style="width:119px;">UFC910024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultra-4 centrifugal filter units</td>
			<td style="width:148px;">Millipore</td>
			<td style="width:119px;">UFC810024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultra-0.5 centrifugal filter units</td>
			<td style="width:148px;">Millipore</td>
			<td style="width:119px;">UFC505024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Optima XE-90 Ultracentrifuge</td>
			<td style="width:148px;">Beckman Coulter</td>
			<td>A94471</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mini-PROTEAN Tetra Cell</td>
			<td style="width:148px;">Bio-Rad</td>
			<td>1658004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mini-PROTEAN precast gel</td>
			<td style="width:148px;">Bio-Rad</td>
			<td>4561084</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T100 Thermal Cycler</td>
			<td style="width:148px;">Bio-Rad</td>
			<td>1861096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PolyJet transfection reagent</td>
			<td style="width:148px;">SignaGen</td>
			<td>SL100688</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pEG BacMam vector</td>
			<td style="width:148px;"></td>
			<td style="width:119px;"></td>
			<td>Obtained from the Gouaux lab at Vollum Institute</td>
		</tr>
	</tbody>
</table>

expression and purification of mammalian bestrophin ion channels

Il genoma umano codifica quattro bestrophin paralogs, vale a dire BEST1, BEST2, BEST3 e BEST4. BEST1, codificata dal gene BEST1 , è Ca2 +-attivato Cl– canale (CaCC) espressi principalmente nell'epitelio retinico del pigmento (RPE). Il significato fisiologico e patologico del BEST1 è evidenziato dal fatto che oltre 200 mutazioni distinte del gene BEST1 sono state collegate geneticamente a uno spettro di almeno cinque disturbi degenerativi della retina, come migliori vitelliforme distrofia maculare (malattia di Best). Pertanto, comprendere la biofisica dei canali di bestrophin a livello di singola molecola contiene un enorme significato. Tuttavia, ottenere canali ionici dei mammiferi purificata è spesso un compito impegnativo. Qui, segnaliamo un protocollo per l'espressione delle proteine di mammiferi bestrophin con il sistema di trasferimento del gene baculovirus BacMam e loro purificazione di affinità e la cromatografia di esclusione dimensionale. Le proteine purificate hanno il potenziale per essere utilizzato nelle successive analisi funzionali e strutturali, quali registrazioni elettrofisiologiche in lipidici e cristallografia. D'importanza, questa pipeline può essere adattata per studiare le funzioni e le strutture di altri canali ionici.

L'intensità di fluorescenza transfected transitoriamente di cellule HEK293 adesiva (Figura 1A) è un buon indicatore per il livello di espressione della proteina proiettata in cellule HEK293-F in sospensione (Figura 1B). Se la proteina dell'obiettivo non è ben espressa o mis-è localizzata nelle cellule HEK293 dopo trasfezione transiente, è consigliabile, è consigliabile modificare il costrutto di espressione (ad es....

Watch this Scientific Journal Video about Expression and Purification of Mammalian Bestrophin Ion Channels at JoVE.com

Expression and Purification of Mammalian Bestrophin Ion Channels

Espressione e purificazione dei canali ionici di mammiferi Bestrophin

La purificazione dei canali ionici è spesso provocatorio, ma una volta raggiunto, potenzialmente può permettere le indagini in vitro delle funzioni e delle strutture dei canali. Qui, descriviamo le procedure graduale per l'espressione e purificazione delle proteine di mammiferi bestrophin, una famiglia di Ca2 +-attivato canali Cl– .

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

expression-and-purification-of-mammalian-bestrophin-ion-channels

Research

JoVE Journal

Biochemistry

8.3K Views.  University of Rochester, School of Medicine and Dentistry. La purificazione dei canali ionici è spesso provocatorio, ma una volta raggiunto, potenzialmente può permettere le indagini in vitro delle funzioni e delle strutture dei canali. Qui, descriviamo le procedure graduale per l'espressione e purificazione delle proteine di mammiferi bestrophin, una famiglia di Ca2 +-attivato canali Cl– .

Video: Expression and Purification of Mammalian Bestrophin Ion Channels

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 &#197; resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

Thermostabilization, Espressione, purificazione e cristallizzazione della umana trasportatore della serotonina associato a<em&gt; S</em&gt; -citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

Ricerca

Biochimica

Expression, Purification, and Antimicrobial Activity of S100A12

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some S100 proteins have the capacity to bind transition metals with high affinity and effectively sequester them away from invading microbial pathogens in a process that is termed "nutritional immunity". S100A12 (EN-RAGE) binds both zinc and copper and is highly abundant in innate immune cells such as macrophages and neutrophils. We report a refined method for the expression, enrichment and purification of S100A12 in its active, metal-binding configuration. Utilization of this protein in bacterial growth and viability analyses reveals that S100A12 has antimicrobial activity against the bacterial pathogen, Helicobacter pylori. The antimicrobial activity is predicated on the zinc-binding activity of S100A12, which chelates nutrient zinc, thereby starving H. pylori which requires zinc for growth and proliferation.

Here, we present a method to express and purify S100A12 (calgranulin C). We describe a protocol to measure its antimicrobial activity against the human pathogen H. pylori.

Espressione, purificazione e attività antimicrobica di S100A12

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some ...

Measurement of Ion Concentration in the Unstirred Boundary Layer with Open Patch-Clamp Pipette: Implications in Control of Ion Channels by Fluid Flow

Fluid flow is an important environmental stimulus that controls many physiological and pathological processes, such as fluid flow-induced vasodilation. Although the molecular mechanisms for the biological responses to fluid flow/shear force are not fully understood, fluid flow-mediated regulation of ion channel gating may contribute critically. Therefore, fluid flow/shear force sensitivity of ion channels has been studied using the patch-clamp technique. However, depending on the experimental protocol, the outcomes and interpretation of data can be erroneous. Here, we present experimental and theoretical evidence for fluid flow-related errors and provide methods for estimating, preventing, and correcting these errors. Changes in junction potential between the Ag/AgCl reference electrode and bathing fluid were measured with an open pipette filled with 3 M KCl. Fluid flow could then shift the liquid/metal junction potential to approximately 7 mV. Conversely, by measuring the voltage shift induced by fluid flow, we estimated the ion concentration in the unstirred boundary layer. In the static condition, the real ion concentrations adjacent to the Ag/AgCl reference electrode or ion channel inlet at the cell-membrane surface can reach as low as approximately 30% of that in the flow condition. Placing an agarose 3 M KCl bridge between the bathing fluid and reference electrode may have prevented this problem of junction potential shifting. However, the unstirred layer effect adjacent to the cell membrane surface could not be fixed in this way. Here, we provide a method for measuring real ion concentrations in the unstirred boundary layer with an open patch-clamp pipette, emphasizing the importance of using an agarose salt-bridge while studying fluid flow-induced regulation of ion currents. Therefore, this novel approach, which takes into consideration the real concentrations of ions in the unstirred boundary layer, may provide useful insight on the experimental design and data interpretation related to fluid shear stress regulation of ion channels.

Mechanosensitive ion channels are often studied in terms of fluid flow/shear force sensitivity with patch-clamp recording. However, depending on the experimental protocol, the outcome on fluid flow-regulations of ion channels can be erroneous. Here, we provide methods for preventing and correcting such errors with a theoretical basis.

Misurazione della concentrazione di ioni nello strato limite Unstirred con pipetta aperta Patch-Clamp: implicazioni nel controllo dei canali ionici dal liquido di flusso

Fluid flow is an important environmental stimulus that controls many physiological and pathological processes, such as fluid flow-induced vasodilation. ...

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium homeostasis, particularly store-operated calcium entry, which is critical to maintaining proper function of synaptic vesicle release and intracellular signaling pathways. Accordingly, TRPC channels have been implicated in a variety of human diseases including cardiovascular disorders such as cardiac hypertrophy, neurodegenerative disorders such as Parkinson&#39;s disease, and neurologic disorders such as spinocerebellar ataxia. Therefore, TRPC channels represent a potential pharmacologic target in human diseases. However, the molecular mechanisms of gating in these channels are still unclear. The difficulty in obtaining large quantities of stable, homogeneous, and purified protein has been a limiting factor in structure determination studies, particularly for mammalian membrane proteins such as the TRPC ion channels. Here, we present a protocol for the large-scale expression of mammalian ion channel membrane proteins using a modified baculovirus gene transfer system and the purification of these proteins by affinity and size-exclusion chromatography. We further present a protocol to collect single-particle cryo-electron microscopy images from purified protein and to use these images to determine the protein structure. Structure determination is a powerful method for understanding the mechanisms of gating and function in ion channels.

This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

Espressione e purificazione di TRPC3 la scanalatura del catione del lipido sensibili umani per determinazione strutturale di singola particella cRIO-microscopia elettronica

Transient receptor potential channels (TRPCs) of the canonical TRP subfamily are nonselective cation channels that play an essential role in calcium ...

Expression, Solubilization, and Purification of Eukaryotic Borate Transporters

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also known as Band 3), the most abundant membrane protein in the red blood cells. The SLC4 family is homologous with borate transporters, which have been characterized in plants and fungi. It remains a significant technical challenge to express and purify membrane transport proteins to homogeneity in quantities suitable for&#160;structural or functional studies. Here we describe detailed procedures for the overexpression of borate transporters in Saccharomyces cerevisiae, isolation of yeast membranes, solubilization of protein by detergent, and purification of borate transporter homologs from S. cerevisiae, Arabidopsis thaliana, and Oryza sativa. We also detail a glutaraldehyde cross-linking experiment to assay multimerization of homomeric transporters. Our generalized procedures can be applied to all three proteins and have been optimized for efficacy. Many of the strategies developed here can be utilized for the study of other challenging membrane proteins.

Here we present a protocol to express, solubilize, and purify several eukaryotic borate transporters with homology to the SLC4 transporter family using yeast. We also describe a chemical cross-linking assay to assess the purified homomeric proteins for multimeric assembly. These protocols can be adapted for other challenging membrane proteins.

Espressione, solubilizzazione e purificazione dei trasportatori eucariotiche borato

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also ...

Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, fluorophores are prone to loss of signal via photobleaching by dissolved oxygen (O2). To prevent photobleaching and extend the fluorophore lifetime, oxygen scavenging systems (OSS) are employed to reduce O2. Commercially available OSS may be contaminated by nucleases that damage or degrade nucleic acids, confounding interpretation of experimental results. Here we detail a protocol for the expression and purification of highly active Pseudomonas putida protocatechuate-3,4-dioxygenase (PCD) with no detectable nuclease contamination. PCD can efficiently remove reactive O2 species by conversion of the substrate protocatechuic acid (PCA) to 3-carboxy-cis,cis-muconic acid. This method can be used in any aqueous system where O2 plays a detrimental role in data acquisition. This method is effective in producing highly active, nuclease free PCD in comparison with commercially available PCD.

Protocatechuate 3,4-dioxygenase (PCD) can enzymatically remove free diatomic oxygen from an aqueous system using its substrate protocatechuic acid (PCA). This protocol describes the expression, purification, and activity analysis of this oxygen scavenging enzyme.

Espressione e purificazione di Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Diossigenasi

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, ...

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

Espressione, purificazione, cristallizzazione e saggi enzimatici di Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of methods. Here, we describe two important approaches used for this purpose. First, we describe how to enrich tissues and cells with cholesterol using cyclodextrin saturated with cholesterol using cerebral arteries (tissues) and hippocampal neurons (cells) as examples. This approach can be used for any type of tissue, cells, or cell lines. An alternative approach for cholesterol enrichment involves the use of low-density lipoprotein (LDL). The advantage of this approach is that it uses part of the natural cholesterol homeostasis machinery of the cell. However, whereas the cyclodextrin approach can be applied to enrich any cell type of interest with cholesterol, the LDL approach is limited to cells that express LDL receptors (e.g., liver cells, bone marrow-derived cells such as blood leukocytes and tissue macrophages), and the level of enrichment depends on the concentration and the mobility of the LDL receptor. Furthermore, LDL particles include other lipids, so cholesterol delivery is nonspecific. Second, we describe how to enrich Xenopus oocytes with cholesterol using a phospholipid-based dispersion (i.e., liposomes) that includes cholesterol. Xenopus oocytes constitute a popular heterologous expression system used for studying cell and protein function. For both the cyclodextrin-based cholesterol enrichment approach of mammalian tissue (cerebral arteries) and for the phospholipid-based cholesterol enrichment approach of Xenopus oocytes, we demonstrate that cholesterol levels reach a maximum following 5 min of incubation. This level of cholesterol remains constant during extended periods of incubation (e.g., 60 min). Together, these data provide the basis for optimized temporal conditions for cholesterol enrichment of tissues, cells, and Xenopus oocytes for functional studies aimed at interrogating the impact of cholesterol enrichment.

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Two methods of cholesterol enrichment are presented: the application of cyclodextrin saturated with cholesterol to enrich mammalian tissues and cells, and the use of cholesterol-enriched phospholipid-based dispersions (liposomes) to enrich Xenopus oocytes. These methods are instrumental for determining the impact of elevated cholesterol levels in molecular, cellular, and organ function.

Arricchimento dei tessuti mammiferi e Xenopus Oocytes con colesterolo

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of ...

PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase

Structural studies with tryptophan synthase (TS) bienzyme complex (&#945;2&#946;2 TS) from Salmonella typhimurium have been performed to better understand its catalytic mechanism, allosteric behavior, and details of the enzymatic transformation of substrate to product in PLP-dependent enzymes. In this work, a novel expression system to produce the isolated &#945;- and isolated &#946;-subunit allowed the purification of high amounts of pure subunits and &#945;2&#946;2 StTS complex from the isolated subunits within 2 days. Purification was carried out by affinity chromatography followed by cleavage of the affinity tag, ammonium sulfate precipitation, and size exclusion chromatography (SEC). To better understand the role of key residues at the enzyme &#946;-site, site-direct mutagenesis was performed in prior structural studies. Another protocol was created to purify the wild type and mutant &#945;2&#946;2 StTS complexes. A simple, fast and efficient protocol using ammonium sulfate fractionation and SEC allowed purification of &#945;2&#946;2 StTS complex in a single day. Both purification protocols described in this work have considerable advantages when compared with previous protocols to purify the same complex using PEG 8000 and spermine to crystalize the &#945;2&#946;2 StTS complex along the purification protocol. Crystallization of wild type and some mutant forms occurs under slightly different conditions, impairing the purification of some mutants using PEG 8000 and spermine. To prepare crystals suitable for x-ray crystallographic studies several efforts were made to optimize crystallization, crystal quality and cryoprotection. The methods presented here should be generally applicable for purification of tryptophan synthase subunits and wild type and mutant &#945;2&#946;2 StTS complexes.

This article presents a series of consecutive methods for the expression and purification of Salmonella typhimurium tryptophan synthase comp this protocol a rapid system to purify the protein complex in a day. Covered methods are site-directed mutagenesis, protein expression in Escherichia coli, affinity chromatography, gel filtration chromatography, and crystallization.

Mutagenesi PCR, clonazione, espressione, protocolli di purificazione rapida delle proteine e cristallizzazione del tipo selvatico e delle forme mutanti di triptofano sintasi

Structural studies with tryptophan synthase (TS) bienzyme complex (&#945;2&#946;2 TS) from Salmonella typhimurium have been performed to better understand ...

Ganglioside Extraction, Purification and Profiling

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially abundant in the brain. Expressed primarily on the outer leaflet of the plasma membranes of cells, they modulate the activities of cell surface proteins via lateral association, act as receptors in cell-cell interactions and are targets for pathogens and toxins. Genetic dysregulation of ganglioside biosynthesis in humans results in severe congenital nervous system disorders. Because of their amphipathic nature, extraction, purification, and analysis of gangliosides require techniques that have been optimized by many investigators in the 80 years since their discovery. Here, we describe bench-level methods for the extraction, purification, and preliminary qualitative and quantitative analyses of major gangliosides from tissues and cells that can be completed in a few hours. We also describe methods for larger scale isolation and purification of major ganglioside species from brain. Together, these methods provide analytical and preparative scale access to this class of bioactive molecules.

Gangliosides are sialic acid-bearing glycosphingolipids that are particularly abundant in the brain. Their amphipathic nature requires organic/aqueous extraction and purification techniques to ensure optimal recovery and accurate analyses. This article provides overviews of analytic and preparative scale ganglioside extraction, purification, and thin layer chromatography analysis.

Estrazione, purificazione e profilatura del ganglioside

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially ...