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

Dette arbeidet ble finansiert av Israels vitenskapsstiftelsens senter for forskningsekspertis (I-CORE) i strukturell cellebiologi (1775/12) og Israels vitenskapsstiftelse gir 1721/16 og 2338/16 (YH) og 825/14 (DK) ). Støtten fra Fields Estate Foundation til DK er høyt verdsatt. Dette arbeidet ble utført av Ilan Edri og Michal Goldenberg i delvis oppfyllelse av MD-avhandlingskravene til Sackler-fakultetet, Tel Aviv-universitetet.

1 . Kloning av cis -PT for overekspresjon i E. coli <ol><li> Hent pET-32b ekspresjonsvektor, designet for kloning og ekspansjon på høyt nivå av proteinsekvenser fusjonert med 109aa tioredoksin (TRX) protein 17 og E. coli kodonoptimalisert 18 kodende sekvens av full lengde cis -PT. </li><li> Pass på å ha et TEV-protease (spaltningsbrenselprotease) spaltningssted (ENLYFQ / G, hvor &quot;/&quot; indikerer spaltningspunktet) <sup clas...

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G., Haitin, Y., Camp, S. S., Black, K. D., Zagotta, W. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanism+for+3:1+subunit+stoichiometry+of+rod+cyclic+nucleotide-gated+ion+channels.">Molecular mechanism for 3:1 subunit stoichiometry of rod cyclic nucleotide-gated ion channels.</a> Nat Commun. 2, 457 (2011).</li><li>Chang, S. Y., Tsai, P. C., Tseng, C. S., Liang, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refolding+and+characterization+of+a+yeast+dehydrodolichyl+diphosphate+synthase+overexpressed+in+Escherichia+coli.">Refolding and characterization of a yeast dehydrodolichyl diphosphate synthase overexpressed in Escherichia coli.</a> Protein Expr Purif. 23 (3), 432-439 (2001).</li><li>Chang, S. Y., Ko, T. P., Liang, P. H., Wang, A. 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Protokollen beskrevet her for rensing av funksjonelle humane DHDDS i E. coli- celler er enkelt og effektivt, slik at man kan overexpresse og rense proteinet i 3 - 4 dager når en egnet konstruksjon er tilgjengelig. Slike protokoller for proteinrensing er av spesiell betydning gitt gjennombruddene i genom-sekvensering, som ga en mengde informasjon om genetikken til mange sykdommer 18 , hvorved det kreves utvikling av høy-gjennomstrømmingsmetoder for å karakterisere patogene mekanismer ...

Prenyltransferaser er en gruppe enzymer som katalyserer kjedeforlengelse av allylldifosfat ved anvendelse av isopentenyl-difosfat (IPP) via flere kondensasjonsreaksjoner 1 , 2 . Z-type enzymer katalyserer dannelsen av cis- dobbeltbindinger fra kondensasjonsreaksjonen, mens E-type enzymer katalyserer trans- dobbeltbindingsdannelse 3 . cis -Prenyltransferases (cis -Pt, Z-type enzymer) er klassisk klassifisert i henhold til deres produkt kjedelengde inn i kortkjedede (C-15), middels kjedelengde (50-55 C), og langkjedede (C 70-120 ) 4 . DHDDS (dehydrodolichyldifosfat-syntase) er en eukaryotisk langkjedet cis -PT som katalyserer kjedeforlenging av farnesyldifosfat (FPP, et allylldifosfat) via flere kondensasjoner med isopentenyldifosfat (IPP) 1, 5 , 6 . Dette resulterer i dannelsen av dehydrodolichyldifosfat, et C 55-100 polyprenyldifosfat som tjener som en forløper for dolichylpyrofosfat, glykosylbærermolekylet involvert i N-koblet proteinglykosylering 1 . Blant Ashkenazi-jøder, resulterer en missens ikke-konservativ mutasjon (K42E) i DHDDS i autosomal recessiv retinitt pigmentosa 7 , 8 . Derfor ble den foreliggende protokollen utviklet for å erverve renset DHDDS egnet for mekanistiske studier. Escherichia coli anses som den mest praktiske og kostnadseffektive verten for rekombinant proteinuttrykk, og er derfor også den mest brukte verten. Men når man forsøker å heterologt overexpresse proteiner i E. coli , bør proteinspesifikke hensyn gjøres. Skaffe riktig foldet, aktivE rekombinante proteiner fra E. coli , er ikke en enkel sak på grunn av de forskjellige egenskapene til forskjellige proteiner. Tallrike tilnærminger har blitt utviklet for å overvinne disse hindrene. Her ble bruken av proteinfusion, optimaliserte kulturbetingelser og kodonoptimalisering benyttet for å tillate overekspresjon og rensing av funksjonelt aktive humane DHDDS i E. coli . Av tone, et tidligere forsøk på å overuttrykker gjær cis -Pt uten proteinfusjonen var mislykket på grunn av fullstendig uoppløselighet selv i nærvær av vaskemiddel 12. Den beskrevne protokollen er enkel, kostnadseffektiv, tidsbesparende og tillater at man oppnår DHDDS-preparater som er egnet for mekanistiske studier. Gitt homologen av cis -PT blant forskjellige arter, foreslår vi at denne protokollen kan brukes for annen eukaryotisk cis -PT også.

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			<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

Prenyltransferaser (PT) er en gruppe enzymer som katalyserer kjedeforlengelse av allylldifosfat ved anvendelse av isopentenyl-difosfat (IPP) via flere kondensasjonsreaksjoner. DHDDS (dehydrodolichyldifosfat-syntase) er en eukaryotisk langkjedet cis -PT (danner cis- dobbeltbindinger fra kondensasjonsreaksjonen) som katalyserer kjedeforlenging av farnesyldifosfat (FPP, et allylldifosfat) via flere kondensasjoner med isopentenyldifosfat (IPP). DHDDS er av biomedisinsk betydning, da en ikke-konservativ mutasjon (K42E) i enzymet resulterer i retinitt pigmentosa , som i siste instans fører til blindhet. Derfor ble den foreliggende protokollen utviklet for å erverve store mengder renset DHDDS, egnet for mekanistiske studier. Her ble bruken av proteinfusjon, optimaliserte kulturbetingelser og kodonoptimalisering benyttet for å tillate overekspresjon og rensing av funksjonelt aktive humane DHDDS i <em&gt; E. Coli. Den beskrevne protokollen er enkel, kostnadseffektiv og tidsbesparende. Homologien til cis -PT blant forskjellige arter antyder at denne protokollen kan anvendes for annen eukaryotisk cis -PT, så vel som de som er involvert i syntetisk syntese.

Generell oversikt over konstruksjonen anvendt her og renseprosessen er vist i figur 1 . Prøver oppnådd ved hvert rensingstrinn er vist i figur 2 . Denne SDS-PAGE-analysen viser trinnvis rensing av DHDDS, noe som resulterer i et høyt renset produkt. Figur 3 viser resultatene av analytisk SEC av det rensede enzymet, og avslører at proteinet kun blir observert som en homodimer. <strong class="xfig"...

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

En enkel protokoll for overekspresjon og rensing av kodonoptimalisert human cis- fenyltransferase under ikke-denaturerende betingelser fra Escherichia Coli , er beskrevet, sammen med en enzymatisk aktivitetsanalyse. Denne protokollen kan generaliseres for produksjon av andre cis- prenyltransferase proteiner i kvantitet og kvalitet egnet for mekanistiske studier.

Overekspresjon og rensing av menneske<em&gt; Cis</em&gt; -prenyltransferase i<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 ...

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