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 arbeidet ble finansiert av Human Frontier Science Program stipend RGP0024.

1. DNA Mal Forberedelse og in vitro Transkripsjon <ol><li> Genet av interesse er klonet i alle T7 og / eller f.eks SP6 promoter basert plasmid. For å oppnå RNCs av interesse, er C-terminus av målet polypeptid utvides ved å legge arrest induserende sekvens av SecM FXXXXWIXXXXGIRAGP 7. For å sikre at polypeptid fragment av interesse vil extrude ut av ribosomal tunnelen, har C-terminalen del av målet proteinet skal forlenges med minst 30 aminosyrer 12-14. En fleksibel Glycine-Serine r...

<ol>
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For reproduserbare resultater, kvalitet og konsentrasjon av komponentene som brukes for in vitro transkripsjon og oversettelse er kritisk. Vi har brukt kommersielt tilgjengelige in vitro transkripsjon og oversettelse ekstrakter og de ​​gir effektive og reproduserbare resultater, hvis håndteres forsiktig. Kvalitet av mRNA kan påvirke oversettelsen, så det er av største betydning for å teste integriteten til mRNA før du bruker den for in vitro oversettelse. Inkubasjonstiden for</em...

<table border="1"><tbody><tr><td> Navn på reagens / Kit </td><td> Firma </td><td> Katalognummer </td></tr><tr><td> MEGAscript T7 høy kapasitet 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> Ribonuclease Inhibitor </td><td> Invitrogen </td><td> 15518012 </td></tr><tr><td> Trans [35 S]-Label </td><td> MP Biomedicals </td><td> 0151006 </td></tr><tr><td> Amicon Ultra-4 sentrifugalfilter Unit </td><td> Millipore </td><td> UFC801008 </td></tr><tr><td> Lagring fosfor autoradiografi </td><td> GE Healthcare </td><td> Typhoon 9410 variabel modus imager </td></tr><tr><td> Tettshetsgradient fraksjonering Systems </td><td> Teledyne Isco, Inc. </td><td> ISCO Programmerbar tettshetsgradient System </td></tr><tr><td> Sukrose Gradient Centrifugation </td><td> Beckman Coulter </td><td> Optima L-90 K preparativ Ultrasentrifuger </td></tr></tbody></table>

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

Omfattende forskning har gitt rikelig med bevis som tyder på at protein folding i cellen er en co-translasjonell prosess 1-5. Imidlertid er den nøyaktige bane som polypeptid kjeden følger under co-translasjonell folding å oppnå sin funksjonelle form fortsatt en gåte. For å forstå denne prosessen og for å fastslå den eksakte konformasjon av co-translasjonelle folding mellomprodukter, er det viktig å utvikle teknikker som tillater isolering av RNCs bærer begynnende kjeder av forhåndsbestemte størrelser å tillate deres videre strukturell analyse. SecM (sekret monitor) er en 170 aminosyre E. coli protein som regulerer uttrykket av nedstrøms Seca (sekret kjøring) ATPasen i secM-seca operon 6. Nakatogawa og Ito opprinnelig fant at en 17 aminosyre lang sekvens (150-FSTPVWISQA QGIRA G P-166) i C-terminal regionen av SecM protein er tilstrekkelig og nødvendig for åforårsake blokkering av SecM forlengelse ved Gly165, og dermed produsere peptidyl-glycyl-tRNA stabilt bundet til ribosomalt P-området 7-9. Enda viktigere, ble det funnet at denne 17 aminosyre lang sekvens kan smeltet til C-terminus av praktisk talt alle full-lengde og / eller avkortet protein og dermed gir produksjon av RNCs bærer begynnende kjeder av forhåndsbestemte størrelser 7. Så når smeltet eller settes inn i målet protein, produserer SecM drøye sekvens arrestasjonen av polypeptid kjedeforlengelse og genererer stabile RNCs både in vivo i E. coli-celler og in vitro i en celle uten system. Sukrose gradient sentrifugering er videre utnyttes for å isolere RNCs. De isolerte RNCs kan brukes til å analysere strukturelle og funksjonelle trekk ved co-translasjonelle folding mellomprodukter. Nylig har denne teknikken vært brukt til å få innsikt i strukturen i flere ribosome innbundne begynnende kjedene 10,11. Her beskriver vi isolering av storfe Gamma-B Crystallin RNCs smeltet SecM og generert i en in vitro oversettelse system.

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 teknikk som nå rutinemessig brukt til å isolere stabilt innbundne ribosomet begynnende kjeden komplekser (RNCs). Denne teknikken utnytter oppdagelsen av at en 17 aminosyre lang SecM &quot;arrest sekvens&quot; kan stanse oversettelse tøyelighet i en prokaryot (<em&gt; E. coli</em&gt;) System, når de settes inn i (eller smeltet til C-terminus) av nesten alle protein.

Bruke SecM Arrest sekvens som et verktøy for å isolere ribosom Innbundne 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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