Name: expression and purification of mammalian bestrophin ion channels
Uploaded: 2018-08-02T14:00:00
Description: Watch this Scientific Journal Video about Expression and Purification of Mammalian Bestrophin Ion Channels at JoVE.com

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...

<ol>
	<li>Hartzell, H. C., Qu, Z., Yu, K., Xiao, Q., Chien, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+physiology+of+bestrophins:+multifunctional+membrane+proteins+linked+to+best+disease+and+other+retinopathies.">Molecular physiology of bestrophins: multifunctional membrane proteins linked to best disease and other retinopathies.</a> Physiological Review. 88 (2), 639-672 (2008).</li><li>Marmorstein, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bestrophin,+the+product+of+the+Best+vitelliform+macular+dystrophy+gene+(VMD2),+localizes+to+the+basolateral+plasma+membrane+of+the+retinal+pigment+epithelium.">Bestrophin, the product of the Best vitelliform macular dystrophy gene (VMD2), localizes to the basolateral plasma membrane of the retinal pigment epithelium.</a> Proceedings of the National Academy of Sciences of the USA. 97 (23), 12758-12763 (2000).</li><li>Marquardt, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+a+novel+gene,+VMD2,+encoding+a+protein+of+unknown+properties+cause+juvenile-onset+vitelliform+macular+dystrophy+(Best's+disease).">Mutations in a novel gene, VMD2, encoding a protein of unknown properties cause juvenile-onset vitelliform macular dystrophy (Best's disease).</a> Human Molecular Genetics. 7 (9), 1517-1525 (1998).</li><li>Petrukhin, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+gene+responsible+for+Best+macular+dystrophy.">Identification of the gene responsible for Best macular dystrophy.</a> Nature Genetics. 19 (3), 241-247 (1998).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-specific+mutations+impair+BESTROPHIN1's+essential+role+in+mediating+Ca2+-dependent+Cl-+currents+in+human+RPE.">Patient-specific mutations impair BESTROPHIN1's essential role in mediating Ca2+-dependent Cl- currents in human RPE.</a> eLife. 6, (2017).</li><li>Tsunenari, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-function+analysis+of+the+bestrophin+family+of+anion+channels.">Structure-function analysis of the bestrophin family of anion channels.</a> Journal of Biological Chemistry. 278 (42), 41114-41125 (2003).</li><li>Kane Dickson, V., Pedi, L., Long, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+insights+into+the+function+of+a+Ca(2+)-activated+Cl(-)+channel.">Structure and insights into the function of a Ca(2+)-activated Cl(-) channel.</a> Nature. 516 (7530), 213-218 (2014).</li><li>Yang, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+selectivity+in+bestrophin+ion+channels.">Structure and selectivity in bestrophin ion channels.</a> Science. 346 (6207), 355-359 (2014).</li><li>Johnson, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bestrophin+1+and+retinal+disease.">Bestrophin 1 and retinal disease.</a> Progress in Retinal and Eye Research. , (2017).</li><li>Allikmets, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+Best+disease+gene+in+patients+with+age-related+macular+degeneration+and+other+maculopathies.">Evaluation of the Best disease gene in patients with age-related macular degeneration and other maculopathies.</a> Human Genetics. 104 (6), 449-453 (1999).</li><li>Burgess, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biallelic+mutation+of+BEST1+causes+a+distinct+retinopathy+in+humans.">Biallelic mutation of BEST1 causes a distinct retinopathy in humans.</a> American Journal of Human Genetics. 82 (1), 19-31 (2008).</li><li>Davidson, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Missense+mutations+in+a+retinal+pigment+epithelium+protein,+bestrophin-1,+cause+retinitis+pigmentosa.">Missense mutations in a retinal pigment epithelium protein, bestrophin-1, cause retinitis pigmentosa.</a> American Journal of Human Genetics. 85 (5), 581-592 (2009).</li><li>Kramer, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+the+VMD2+gene+are+associated+with+juvenile-onset+vitelliform+macular+dystrophy+(Best+disease)+and+adult+vitelliform+macular+dystrophy+but+not+age-related+macular+degeneration.">Mutations in the VMD2 gene are associated with juvenile-onset vitelliform macular dystrophy (Best disease) and adult vitelliform macular dystrophy but not age-related macular degeneration.</a> European Journal of Human Genetics. 8 (4), 286-292 (2000).</li><li>Yardley, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+of+VMD2+splicing+regulators+cause+nanophthalmos+and+autosomal+dominant+vitreoretinochoroidopathy+(ADVIRC).">Mutations of VMD2 splicing regulators cause nanophthalmos and autosomal dominant vitreoretinochoroidopathy (ADVIRC).</a> Investigative Ophthalmology &amp; Visual Science. 45 (10), 3683-3689 (2004).</li><li>Yang, T., Justus, S., Li, Y., Tsang, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BEST1:+the+Best+Target+for+Gene+and+Cell+Therapies.">BEST1: the Best Target for Gene and Cell Therapies.</a> Molecular Therapy. 23 (12), 1805-1809 (2015).</li><li>Boyce, F. M., Bucher, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculovirus-mediated+gene+transfer+into+mammalian+cells.">Baculovirus-mediated gene transfer into mammalian cells.</a> Proceedings of the National Academy of Sciences of the USA. 93 (6), 2348-2352 (1996).</li><li>Kost, T. A., Condreay, J. P., Jarvis, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baculovirus+as+versatile+vectors+for+protein+expression+in+insect+and+mammalian+cells.">Baculovirus as versatile vectors for protein expression in insect and mammalian cells.</a> Nature Biotechnology. 23 (5), 567-575 (2005).</li><li>Goehring, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+and+large-scale+expression+of+membrane+proteins+in+mammalian+cells+for+structural+studies.">Screening and large-scale expression of membrane proteins in mammalian cells for structural studies.</a> Nature Protocols. 9 (11), 2574-2585 (2014).</li><li>Yang, T., He, L. L., Chen, M., Fang, K., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bio-inspired+voltage-dependent+calcium+channel+blockers.">Bio-inspired voltage-dependent calcium channel blockers.</a> Nature Communications. 4, 2540 (2013).</li><li>Yang, T., Hendrickson, W. A., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preassociated+apocalmodulin+mediates+Ca2+-dependent+sensitization+of+activation+and+inactivation+of+TMEM16A/16B+Ca2+-gated+Cl-+channels.">Preassociated apocalmodulin mediates Ca2+-dependent sensitization of activation and inactivation of TMEM16A/16B Ca2+-gated Cl- channels.</a> Proceedings of the National Academy of Sciences of the USA. 111 (51), 18213-18218 (2014).</li><li>Yang, T., Puckerin, A., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+RGK+GTPases+differentially+use+alpha1-+and+auxiliary+beta-binding-dependent+mechanisms+to+inhibit+CaV1.2/CaV2.2+channels.">Distinct RGK GTPases differentially use alpha1- and auxiliary beta-binding-dependent mechanisms to inhibit CaV1.2/CaV2.2 channels.</a> Public Library of Science One. 7 (5), e37079 (2012).</li><li>Yang, T., Suhail, Y., Dalton, S., Kernan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+molecules+for+inducibly+inactivating+CaV+channels.">Genetically encoded molecules for inducibly inactivating CaV channels.</a> Nature Chemical Biology. 3 (12), 795-804 (2007).</li><li>Yang, T., Xu, X., Kernan, T., Wu, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rem,+a+member+of+the+RGK+GTPases,+inhibits+recombinant+CaV1.2+channels+using+multiple+mechanisms+that+require+distinct+conformations+of+the+GTPase.">Rem, a member of the RGK GTPases, inhibits recombinant CaV1.2 channels using multiple mechanisms that require distinct conformations of the GTPase.</a> Journal of Physiology. 588 (Pt 10), 1665-1681 (2010).</li><li>Kawate, T., Gouaux, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-detection+size-exclusion+chromatography+for+precrystallization+screening+of+integral+membrane+proteins.">Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins.</a> Structure. 14 (4), 673-681 (2006).</li><li>Schmidt, C., Urlaub, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+cryo-electron+microscopy+(cryo-EM)+and+cross-linking+mass+spectrometry+(CX-MS)+for+structural+elucidation+of+large+protein+assemblies.">Combining cryo-electron microscopy (cryo-EM) and cross-linking mass spectrometry (CX-MS) for structural elucidation of large protein assemblies.</a> Currents Opinions in Structural Biology. 46, 157-168 (2017).</li><li>Sun, W., Zheng, W., Simeonov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+discovery+and+development+for+rare+genetic+disorders.">Drug discovery and development for rare genetic disorders.</a> American Journal of Medical Genetics Part A. 173 (9), 2307-2322 (2017).</li></ol>

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....

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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- ...

Research

JoVE Journal

Biochemistry

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

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

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

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Ricerca

Biochimica

Expression, Purification, and Antimicrobial Activity of S100A12

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

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

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

Espressione, solubilizzazione e purificazione dei trasportatori eucariotiche borato

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

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

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

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

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

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Ganglioside Extraction, Purification and Profiling

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 ...

Efficient and Scalable Production of Full-length Human Huntingtin Variants in Mammalian Cells using a Transient Expression System

Full-length huntingtin (FL HTT) is a large (aa 1-3,144), ubiquitously expressed, polyglutamine (polyQ)-containing protein with a mass of approximately 350 ...