Name: overexpression and purification of human cis-prenyltransferase in escherichia coli
Uploaded: 2017-08-03T15:00:00
Description: Watch this Scientific Journal Video about Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli at JoVE.com

Dit werk werd gefinancierd door het Israëlic Science Foundation&#39;s Center for Research Excellence (I-CORE) in de Structurele Celbiologie (1775/12) en de Israëlic Science Foundation verleent 1721/16 en 2338/16 (YH) en 825/14 (DK ). De steun van de Fields Estate Foundation naar DK wordt zeer gewaardeerd. Dit werk werd uitgevoerd door Ilan Edri en Michal Goldenberg in gedeeltelijke vervulling van de MD thesis eisen van de Sackler Faculteit Geneeskunde, Tel Aviv University.

1. Klonen van cis -PT voor overexpressie in E. coli <ol><li> Verkrijg pET-32b expressievector, ontworpen voor klonen en expressie op hoog niveau van eiwitsequenties gefuseerd met het 109aa thioredoxine (TRX) eiwit 17 , en E. coli codon-geoptimaliseerde 18 coderende sequentie van cis -PT met volledige lengte. </li><li> Zorg ervoor dat een TEV-protease-splitsingsplaats (tabak etsvirus protease) is gesplitst (ENLYFQ / G, waar &quot;/&quot...

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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=Crystal+structures+of+undecaprenyl+pyrophosphate+synthase+in+complex+with+magnesium,+isopentenyl+pyrophosphate,+and+farnesyl+thiopyrophosphate:+roles+of+the+metal+ion+and+conserved+residues+in+catalysis.">Crystal structures of undecaprenyl pyrophosphate synthase in complex with magnesium, isopentenyl pyrophosphate, and farnesyl thiopyrophosphate: roles of the metal ion and conserved residues in catalysis.</a> J Biol Chem. 280 (21), 20762-20774 (2005).</li><li>Collins, F. S., Green, E. D., Guttmacher, A. E., Guyer, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+vision+for+the+future+of+genomics+research.">A vision for the future of genomics research.</a> Nature. 422 (6934), 835-847 (2003).</li><li>Freeze, H. 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Het protocol dat hier wordt beschreven voor zuivering van functionele humane DHDDS in E. coli cellen is eenvoudig en efficiënt, waardoor men het eiwit over 3-4 dagen kan uitdrukken en zuiveren zodra een geschikt construct beschikbaar is. Dergelijke protocollen voor eiwitzuivering zijn van bijzondere betekenis gegeven door de doorbraken in genoomsequentiebepaling, die veel informatie verschaft over de genetica van vele ziekten 18 , waardoor de ontwikkeling van high-throughputmethoden ver...

Prenyltransferasen zijn een groep enzymen die de kettingverlenging van allylischifosfaat met behulp van isopentenyldifosfaat (IPP) katalyseren via meerdere condensatie reacties 1 , 2 . Z-type enzymen katalyseren de vorming van cis dubbele bindingen uit de condensatiereactie, terwijl E-type enzymen trans- dubbelbindingsvorming 3 katalyseren. cis -Prenyltransferases (cis -PT, Z-type enzymen) zijn klassiek geclassificeerd volgens hun product ketenlengte in korte keten (C15), middellange-keten (50-55) en lange-keten (70-120 ) 4 . DHDDS (dehydrodolichyl difosfaat synthase) een eukaryotische langketenige cis -PT die ketenverlenging katalyseert van farnesyl difosfaat (FPP, een allylisch difosfaat) via meerdere condensaties met isopentenyldifosfaat (IPP) 1, 5 , 6 . Dit resulteert in de vorming van dehydrodolichyldifosfaat, een C 55-100 polyprenyldifosfaat dat als voorloper voor dolichylpyrofosfaat, de glycosyldragermolecuul betrokken bij N-gekoppelde eiwitglycosylering 1 dient . Bij Ashkenazi-joden resulteert een missense niet-conservatieve mutatie (K42E) in DHDDS autosomale recessieve retinitis pigmentosa 7 , 8 . Daarom is het onderhavige protocol ontwikkeld om zuiverde DHDDS te verkrijgen die geschikt zijn voor mechanistische studies. Escherichia coli wordt beschouwd als de meest geschikte en kosteneffectieve gastheer voor recombinante eiwit expressie, en is daarom ook de meest gebruikte gastheer. Wanneer men echter eiwitten in E. coli heterologisch overexpresseert, moeten eiwitspecifieke overwegingen worden gemaakt. Verkrijgen van goed gevouwen, actiefE recombinante eiwitten uit E. coli , is niet een eenvoudige kwestie door de verschillende eigenschappen van verschillende eiwitten. Er zijn talrijke aanpak ontwikkeld om deze hindernissen te overwinnen. Hierbij werd het gebruik van eiwitfusie, geoptimaliseerde kweekomstandigheden en codonoptimalisatie gebruikt om de overexpressie en zuivering van functioneel actieve humane DHDDS in E. coli toe te staan . Van te voren was een eerdere poging om gist cis -PT te overexpressen zonder eiwitfusie mislukt door volledige onoplosbaarheid zelfs in aanwezigheid van detergent 12 . Het beschreven protocol is eenvoudig, kosteneffectief, tijdbesparend en maakt het mogelijk om DHDDS preparaten te verkrijgen die geschikt zijn voor mechanistische studies. Gezien de homologie van cis -PT tussen verschillende soorten, raden we aan dat dit protocol ook toegepast kan worden op andere eukaryotische cis -PT.

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		<col />
		<col />
		<col />
		<col />
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	<tbody>
		<tr height="21">
			<td height="21" style="width:365px;height:21px;">pET-32b</td>
			<td style="width:100px;">Novagen</td>
			<td style="width:119px;">69016-3</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T7 Express&#160;lysY&#160;Competent&#160;E. coli&#160;(High Efficiency)</td>
			<td style="width:100px;">NEB</td>
			<td>C3010I</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:365px;height:42px;">cOmplete, EDTA-free Protease Inhibitor Cocktail</td>
			<td style="width:100px;">Roche</td>
			<td style="width:119px;">11873580001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">TALON-superflow resin</td>
			<td style="width:100px;">GE Healthcare</td>
			<td>28-9574-99</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">HiPrep 26/10 desalting column&#160;</td>
			<td style="width:100px;">GE Healthcare</td>
			<td>17508701</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">HiLoad 16/60 superdex-200&#160;</td>
			<td style="width:100px;">GE Healthcare</td>
			<td>28989335</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">superdex-200 increase 5/150 GL&#160;</td>
			<td style="width:100px;">GE Healthcare</td>
			<td>28990945</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">14C-Isopentenyl pyrophosphate</td>
			<td style="width:100px;">Perkin-Elmer</td>
			<td>NEC773050UC&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:365px;height:21px;">trans,trans-Farnesyl pyrophosphate</td>
			<td style="width:100px;">Sigma</td>
			<td>44270-10MG</td>
			<td></td>
		</tr>
	</tbody>
</table>

overexpression and purification of human cis-prenyltransferase in escherichia coli

Prenyltransferases (PT) zijn een groep enzymen die de kettingverlenging van allylic difosfaat met behulp van isopentenyldifosfaat (IPP) katalyseren via meerdere condensatie reacties. DHDDS (dehydrodolichyl difosfaat synthase) een eukaryotische langketenige cis -PT (vorming van cis-dubbele bindingen van de condensatiereactie) die ketenverlenging van farnesyl difosfaat (FPP, een allylisch difosfaat) katalyseert via meerdere condensaties met isopentenyldifosfaat (IPP). DHDDS is van biomedisch belang, aangezien een niet-conservatieve mutatie (K42E) in het enzym resulteert in retinitis pigmentosa , wat uiteindelijk tot blindheid leidt. Daarom is het onderhavige protocol ontwikkeld om grote hoeveelheden gezuiverde DHDDS te verkrijgen, geschikt voor mechanistische studies. Hierbij werd het gebruik van eiwitfusie, geoptimaliseerde kweekomstandigheden en codonoptimalisatie gebruikt om de overexpressie en zuivering van functioneel actieve humane DHDDS in <em&gt; E. Coli. Het beschreven protocol is eenvoudig, kosteneffectief en tijdbesparend. De homologie van cis -PT tussen verschillende soorten suggereert dat dit protocol ook toegepast kan worden op andere eukaryotische cis -PT, zoals die welke betrokken zijn bij synthetische synthetische rubber.

Algemeen overzicht van het hier gebruikte constructie en het zuiveringsproces worden getoond in Figuur 1 . De bij elke zuiveringsstap verkregen monsters worden getoond in figuur 2 . Deze SDS-PAGE analyse toont de stapsgewijze zuivering van DHDDS, wat resulteert in een sterk gezuiverd product. Figuur 3 toont de resultaten van analytische SEC van het gezuiverde enzym, waarbij wordt gebleken dat het ei...

Watch this Scientific Journal Video about Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli at JoVE.com

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Een eenvoudig protocol voor overexpressie en zuivering van codon-geoptimaliseerde, humane cis -prenyltransferase, onder niet-denaturerende omstandigheden, van Escherichia Coli , wordt beschreven, samen met een enzymatische activiteitstest. Dit protocol kan worden genegaliseerd voor de productie van andere cis- prenyltransferase eiwitten in hoeveelheid en kwaliteit geschikt voor mechanistische studies.

Overexpressie en zuivering van het menselijk<em&gt; Cis</em&gt; -prenyltransferase in<em&gt; Escherichia coli</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

The overall goal of this protocol is to overexpress and purify codon-optimized human cis-prenyltransferase under non-denaturing conditions and to assay its enzymatic activity. The procedure is demonstrated with the eukaryotic long-chain cis-prenyltransferase dehydrodolichyl diphosphate synthase, or DHDDS. This method can advance our understanding of isoprenoid synthesis and provide key insights into the pathophysiological mechanism of retinitis pigmentosa related to mutations in DHDDS.The main advantages of this technique is that it's simple, cost-effective, and time-saving. It also can be generalized for high-quantity production of other cis-prenyltransferase proteins. Begin this procedure with cloning an expression of human DHDDS, as described in the text protocol.Resuspend the cells in buffer A, one microgram per milliliter of DNase I, and a protease inhibitor mixture. Then, homogenize the resuspended cells using a glass Teflon homogenizer. Disrupt the cells with a Microfluidizer or equivalent at 12, 000 to 15, 000 psi.Next, centrifuge the cell lysate at 40, 000 times g for 45 minutes at four degrees Celsius. Recover the supernatant containing the soluble fraction. Load the supernatant onto a five to 10 millimeter cobalt-immobilized metal affinity chromatography column with 10 millimolar imidazole.After loading into a fast protein liquid chromatography machine, perform a thorough washing of the column with buffer A in 10 millimolar imidazole in order to reduce non-specific protein binding. Begin the FPLC program. Elute the overexpressed proteins with buffer A supplemented with 250 millimolar imidazole.Following elution, remove the imidazole using 53 milliliters of a preparative desalting column equilibrated with buffer A.Then, add hexahistidine-tagged TEV protease to the eluted proteins to remove their hexahistidine-tagged thioredoxin DHDDS fusion. Incubate the mixture at four degrees Celsius overnight. Next, load the cleaved proteins onto a cobalt-immobilized metal affinity chromatography column equilibrated with buffer A supplemented with five millimolar imidazole.Collect the flowthrough to remove the cleaved hexahistidine-tagged thioredoxin and TEV protease. Concentrate the flowthrough to between three and four milliliters using a 30 kilodalton molecular weight cut off centrifugal filter. Then, load the concentrated protein onto a Superdex 200 size exclusion chromatography column equilibrated with buffer A for final purification.Place the samples into a 96-well rack in a fraction collector. The protein elutes as a dimer. Select relevant fractions by comparing the elution profile to a column calibration curve.At this point, assess the purity of the preparation using SDS-PAGE. Finally, determine the protein concentration needed for downstream experimentation and flash freeze aliquots of purified concentrated proteins in liquid nitrogen. Store the aliquots at minus 80 degrees Celsius until use.To ensure the ability of the protein to endure freeze-thaw cycles, thaw an aliquot of the frozen proteins. Centrifuge the aliquot at 21, 000 times g for 10 minutes to remove insoluble aggregates. Following centrifugation, collect the supernatant.Next, load the proteins onto a Superdex 200 analytical column pre-equilibrated with TNXB using an ultra performance liquid chromatography system. Monitor the tryptophan fluorescence to detect the protein's elution profile. Be sure to have a valid calibration curve for molecular weight assessment, which is available via the SEC column manufacturers.To ensure the activity of the purified protein, first mix five micromolar of purified DHDDS with 10 micromolar FPP and 50 micromolar C14 IPP. Initiate the reaction in buffer A with 0.5 millimolar magnesium chloride at 22 to 30 degrees Celsius. Withdraw 15 microliter samples from the reaction at zero, two, four, and six hours following immediate quenching of the reaction by the addition of 15 microliters of buffer A supplemented with 20 millimolar EDTA.Then, add one milliliter of water-saturated 1-butanol and vortex thoroughly to extract the reaction products. The protocol can be stopped here, and the samples can be kept for later reading. Next, add the scintillation cocktail to the samples.Using a scintillation counter, quantitate reaction products in the butanol phase encompassing C14 together with radioactivity of 15 microliters of the reaction, representing the total radioactivity. Finally, calculate the net C14 IPP incorporation at each time point by calculating the percent utilized from the total C14 IPP concentrations and plot the results as a function of time. An increase in the net C14 IPP incorporation is expected with time.The samples obtained at each purification step are shown here. This SDS-PAGE analysis shows the stepwise purification of DHDDS, resulting in a highly-purified product. This chromatogram represents the results of analytical size exclusion chromatography of the purified enzyme, revealing that the protein is only observed as a homodimer.Here, the arrow indicates the void volume. A representative time-dependent activity assay reveals that C14 IPP incorporation clearly rises over six hours, verifying that the purified enzyme is functional. Once mastered, this simple preparation can be done in two to three days, resulting in a highly pure and active enzyme.After watching this video, you should have a good understanding of how to overexpress and purify human cis-prenyltransferase from E.coli and to measure its activity in a time-dependent manner.

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JoVE Journal

Immunology and Infection

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

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Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Detectie van de Enterohemorrhagic Escherichia Coli kolonisatie in lymfkliertest Host door niet-invasieve In Vivo bioluminescentie systeem

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Oprichting van een dichte Transposon invoeging bibliotheek met behulp van bacteriële Woordherkomst en-opbouw in de Enterobacterial stammen zoals Escherichia Coli of Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Een Facile Protocol voor het genereren van Site-Specifically geacetyleerd eiwitten in Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...