Name: using secm arrest sequence as a tool to isolate ribosome bound polypeptides
Uploaded: 2012-06-19T12:00:00
Description: Watch this Scientific Journal Video about Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides at JoVE.com

Dette arbejde blev finansieret af Human Frontier Science Program tilskud RGP0024.

1. DNA-template Fremstilling og in vitro-transskription <ol><li> Genet af interesse klones i enhver T7 og / eller f.eks SP6-promotoren baseret plasmid. Til opnåelse RNC&#39;er af interesse, er C-terminalen af målpolypeptidet forlænget ved tilføjelse arrestationen inducerende sekvens SecM FXXXXWIXXXXGIRAGP 7. For at sikre, at polypeptidfragmentet af interesse vil ekstrudere af den ribosomale tunnelen, den C-terminale del af målproteinet skal udvides med mindst 30 aminosyrer 12-14. En...

<ol>
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For reproducerbare resultater, og koncentration af de komponenter, der anvendes til in vitro transkription og translation er kritiske. Vi har anvendt kommercielt tilgængeligt in vitro transkriptions-og translationselementer ekstrakter og de ​​giver en effektiv og reproducerbare resultater, hvis den håndteres forsigtigt. Kvaliteten af mRNA kan påvirke translation, så er det af yderste vigtighed for at teste integriteten af mRNA&#39;en før brug for in vitro translation...

<table border="1"><tbody><tr><td> Navnet reagens / Kit </td><td> Firma </td><td> Katalog nummer </td></tr><tr><td> MEGAscript T7 Højt udbytte Transcription Kit </td><td> Ambion </td><td> AM1333 </td></tr><tr><td> RTS 100 E.coli HY Kit </td><td> 5 Prime </td><td> 2401100 </td></tr><tr><td> Ribonucleaseinhibitor </td><td> Invitrogen </td><td> 15518012 </td></tr><tr><td> Trans [35S]-Label </td><td> MP Biomedicals </td><td> 0151006 </td></tr><tr><td> Amicon Ultra-4 centrifugalfilterenhed </td><td> Millipore </td><td> UFC801008 </td></tr><tr><td> Opbevaring fosfor autoradiografi </td><td> GE Healthcare </td><td> Typhoon 9410 variabel tilstand imager </td></tr><tr><td> Densitetsgradientfraktionering Systems </td><td> Teledyne Isco, Inc. </td><td> ISCO Programmerbar Density Gradient System </td></tr><tr><td> Saccharosegradient Centrifugation </td><td> Beckman Coulter </td><td> Optima L-90 K Præparativ Ultracentrifuge </td></tr></tbody></table>

using secm arrest sequence as a tool to isolate ribosome bound polypeptides

Omfattende forskning har givet rigelige beviser tyder på, at proteinfoldning i cellen er en co-translationel proces 1-5. Men den nøjagtige vej at polypeptidkæde følger i løbet af co-translationel folde for at opnå den funktionelle form er stadig en gåde. For at forstå denne proces og til at bestemme den nøjagtige konformationen af ​​de co-translationale foldning mellemprodukter, er det vigtigt at udvikle teknikker, der tillader isolering af RNC&#39;er bærer nascente kæder af forudbestemte størrelser, så de yderligere strukturel analyse. SecM (sekretion monitor) er et 170 aminosyre E. coli-protein, der regulerer ekspressionen af den nedstrøms SecA (sekretion kørsel) ATPase i secM-SecA operon 6. Nakatogawa og Ito oprindeligt fundet, at en 17 aminosyre lange sekvens (150-FSTPVWISQA QGIRA G P-166) i den C-terminale region af SecM proteinet er tilstrækkeligt til atforårsage blokering af SecM forlængelse ved Gly165, hvorved der frembringes peptidyl-glycyl-tRNA stabilt bundet til det ribosomale P-site 7-9. Vigtigere, blev det konstateret, at denne 17 aminosyrer lange sekvens kan fusioneres til den C-terminale ende af praktisk talt alle fuld længde og / eller trunkeret protein således tillader fremstillingen af RNC&#39;er bærer nascente kæder forudbestemte størrelser 7. Således, når det er fusioneret eller indsættes i målproteinet, SecM blokering sekvens frembringer standsning af polypeptidet kædeforlængelse og genererer stabile RNC&#39;er både in vivo i E. E. coli celler og in vitro i et celle-frit system. Saccharosegradientcentrifugering yderligere anvendes til at isolere RNC&#39;er. De isolerede RNC&#39;er kan anvendes til at analysere strukturelle og funktionelle træk ved de co-translationelle foldning mellemprodukter. For nylig er denne teknik blevet anvendt til at opnå indsigt i strukturen af adskillige ribosom bundne nascente kæder 10,11. Her beskrives isoleringen af bovine gamma-B crystallin RNC&#39;er fusioneret til SecM og genereret i et in vitro translationssystem.

Watch this Scientific Journal Video about Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides at JoVE.com

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Vi beskriver her en teknik, der nu rutinemæssigt anvendes til at isolere stabilt bundet ribosom vordende kæde-komplekser (RNC&#39;er). Denne teknik udnytter den erkendelse, at en 17 aminosyre langt SecM &quot;arrest sekvens&quot; kan standse translation forlængelse i en prokaryot (<em&gt; E. coli</em&gt;) System, når det indsættes i (eller fusioneret til C-terminus) af praktisk taget hvilket som helst protein.

Brug SecM Arrest Sequence som et redskab til at isolere ribo bundne polypeptider

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

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