Name: a g-quadruplex dna-affinity approach for purification of enzymatically active g4 resolvase1
Uploaded: 2017-03-18T14:00:00
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Vi ønsker å takke våre finansieringskilder, inkludert en generøs gave fra Ware Foundation (til JPV), The National Institutes of HealthGrant T32-CA079448 (til PJS), og Ball State University oppstartsfond (til PJS). Finansiører hadde ingen rolle i studiedesign, datainnsamling og analyse, beslutning om å publisere, eller utarbeidelse av manuskriptet.

1. Utarbeidelse av G4-DNA strukturer til bruk for rensing av rG4R1 (Dannelse av Biotinylated G4-DNA G-quadruplex) <ol><li> Bestill følgende DNA oligomer, heter Z33-Bio, på en 1 umol-skala: 5 &#39;AAA GTG ATG GTG GTG GGG GAA GGA TTC GGA CCT-biotin 3&#39;. Kontroller at biotinrest er i 3&#39;-enden av oligomeren. </li><li> Forbered 10x G4-buffer: 450 mM Tris-HCl pH 8, 25 mM EDTA, og 2.500 mM NaCl. </li><li> Resuspender Z33 oligomeren i 250 ul av vann (slik at oligomeren er konsentrasjonen ~ 2,5 mM). Legg 25 ul av 10 x...

<ol>
	<li>Qin, Y., Hurley, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structures,+folding+patterns,+and+functions+of+intramolecular+DNA+G-quadruplexes+found+in+eukaryotic+promoter+regions.">Structures, folding patterns, and functions of intramolecular DNA G-quadruplexes found in eukaryotic promoter regions.</a> Biochimie. 90 (8), 1149-1171 (2008).</li><li>Stegle, O., Payet, L., Mergny, J. L., MacKay, D. J., Leon, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+and+understanding+the+stability+of+G-quadruplexes.">Predicting and understanding the stability of G-quadruplexes.</a> Bioinformatics. 25 (12), i374-i382 (2009).</li><li>Todd, A. K., Johnston, M., Neidle, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+prevalent+putative+quadruplex+sequence+motifs+in+human+DNA.">Highly prevalent putative quadruplex sequence motifs in human DNA.</a> Nucleic Acids Res. 33 (9), 2901-2907 (2005).</li><li>Huppert, J. L., Balasubramanian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+quadruplexes+in+the+human+genome.">Prevalence of quadruplexes in the human genome.</a> Nucleic Acids Res. 33 (9), 2908-2916 (2005).</li><li>Bedrat, A., Lacroix, L., Mergny, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Re-evaluation+of+G-quadruplex+propensity+with+G4Hunter.">Re-evaluation of G-quadruplex propensity with G4Hunter.</a> Nucleic Acids Res. 44 (4), 1746-1759 (2016).</li><li>Mendoza, O., Bourdoncle, A., Boule, J. B., Brosh, R. M., Mergny, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G-quadruplexes+and+helicases.">G-quadruplexes and helicases.</a> Nucleic Acids Res. 44 (5), 1989-2006 (2016).</li><li>Huppert, J. L., Balasubramanian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G-quadruplexes+in+promoters+throughout+the+human+genome.">G-quadruplexes in promoters throughout the human genome.</a> Nucleic Acids Res. 35 (2), 406-413 (2007).</li><li>Eddy, J., Maizels, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+function+correlates+with+potential+for+G4+DNA+formation+in+the+human+genome.">Gene function correlates with potential for G4 DNA formation in the human genome.</a> Nucleic Acids Res. 34 (14), 3887-3896 (2006).</li><li>Eddy, 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=G4+motifs+correlate+with+promoter-proximal+transcriptional+pausing+in+human+genes.">G4 motifs correlate with promoter-proximal transcriptional pausing in human genes.</a> Nucleic Acids Res. 39 (12), 4975-4983 (2011).</li><li>Vaughn, J. P., 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=The+DEXH+protein+product+of+the+DHX36+gene+is+the+major+source+of+tetramolecular+quadruplex+G4-DNA+resolving+activity+in+HeLa+cell+lysates.">The DEXH protein product of the DHX36 gene is the major source of tetramolecular quadruplex G4-DNA resolving activity in HeLa cell lysates.</a> J Biol Chem. 280 (46), 38117-38120 (2005).</li><li>Booy, E. P., 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=The+RNA+helicase+RHAU+(DHX36)+unwinds+a+G4-quadruplex+in+human+telomerase+RNA+and+promotes+the+formation+of+the+P1+helix+template+boundary.">The RNA helicase RHAU (DHX36) unwinds a G4-quadruplex in human telomerase RNA and promotes the formation of the P1 helix template boundary.</a> Nucleic Acids Res. 40 (9), 4110-4124 (2012).</li><li>Creacy, S. 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=G4+resolvase+1+binds+both+DNA+and+RNA+tetramolecular+quadruplex+with+high+affinity+and+is+the+major+source+of+tetramolecular+quadruplex+G4-DNA+and+G4-RNA+resolving+activity+in+HeLa+cell+lysates.">G4 resolvase 1 binds both DNA and RNA tetramolecular quadruplex with high affinity and is the major source of tetramolecular quadruplex G4-DNA and G4-RNA resolving activity in HeLa cell lysates.</a> J Biol Chem. 283 (50), 34626-34634 (2008).</li><li>Giri, B., 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=G4+resolvase+1+tightly+binds+and+unwinds+unimolecular+G4-DNA.">G4 resolvase 1 tightly binds and unwinds unimolecular G4-DNA.</a> Nucleic Acids Res. 39 (16), 7161-7178 (2011).</li><li>Lattmann, S., Stadler, M. B., Vaughn, J. P., Akman, S. A., Nagamine, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DEAH-box+RNA+helicase+RHAU+binds+an+intramolecular+RNA+G-quadruplex+in+TERC+and+associates+with+telomerase+holoenzyme.">The DEAH-box RNA helicase RHAU binds an intramolecular RNA G-quadruplex in TERC and associates with telomerase holoenzyme.</a> Nucleic Acids Res. 39 (21), 9390-9404 (2011).</li><li>Sexton, A. N., Collins, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+5'+guanosine+tracts+of+human+telomerase+RNA+are+recognized+by+the+G-quadruplex+binding+domain+of+the+RNA+helicase+DHX36+and+function+to+increase+RNA+accumulation.">The 5' guanosine tracts of human telomerase RNA are recognized by the G-quadruplex binding domain of the RNA helicase DHX36 and function to increase RNA accumulation.</a> Mol Cell Biol. 31 (4), 736-743 (2011).</li><li>Smaldino, P. 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=Mutational+Dissection+of+Telomeric+DNA+Binding+Requirements+of+G4+Resolvase+1+Shows+that+G4-Structure+and+Certain+3'-Tail+Sequences+Are+Sufficient+for+Tight+and+Complete+Binding.">Mutational Dissection of Telomeric DNA Binding Requirements of G4 Resolvase 1 Shows that G4-Structure and Certain 3'-Tail Sequences Are Sufficient for Tight and Complete Binding.</a> PLoS One. 10 (7), e0132668 (2015).</li><li>Booy, E. P., 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=The+RNA+helicase+RHAU+(DHX36)+suppresses+expression+of+the+transcription+factor+PITX1.">The RNA helicase RHAU (DHX36) suppresses expression of the transcription factor PITX1.</a> Nucleic Acids Res. 42 (5), 3346-3361 (2014).</li><li>Huang, W., 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=Yin+Yang+1+contains+G-quadruplex+structures+in+its+promoter+and+5'-UTR+and+its+expression+is+modulated+by+G4+resolvase+1.">Yin Yang 1 contains G-quadruplex structures in its promoter and 5'-UTR and its expression is modulated by G4 resolvase 1.</a> Nucleic Acids Res. 40 (3), 1033-1049 (2012).</li><li>Tran, H., Schilling, M., Wirbelauer, C., Hess, D., Nagamine, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facilitation+of+mRNA+deadenylation+and+decay+by+the+exosome-bound,+DExH+protein+RHAU.">Facilitation of mRNA deadenylation and decay by the exosome-bound, DExH protein RHAU.</a> Mol Cell. 13 (1), 101-111 (2004).</li><li>Yoo, J. S., 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=DHX36+enhances+RIG-I+signaling+by+facilitating+PKR-mediated+antiviral+stress+granule+formation.">DHX36 enhances RIG-I signaling by facilitating PKR-mediated antiviral stress granule formation.</a> PLoS Pathog. 10 (3), e1004012 (2014).</li><li>Lai, J. C., 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=The+DEAH-box+helicase+RHAU+is+an+essential+gene+and+critical+for+mouse+hematopoiesis.">The DEAH-box helicase RHAU is an essential gene and critical for mouse hematopoiesis.</a> Blood. 119 (18), 4291-4300 (2012).</li><li>Zhang, Z., 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=DDX1,+DDX21,+and+DHX36+helicases+form+a+complex+with+the+adaptor+molecule+TRIF+to+sense+dsRNA+in+dendritic+cells.">DDX1, DDX21, and DHX36 helicases form a complex with the adaptor molecule TRIF to sense dsRNA in dendritic cells.</a> Immunity. 34 (6), 866-878 (2011).</li><li>Kim, 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=Aspartate-glutamate-alanine-histidine+box+motif+(DEAH)/RNA+helicase+A+helicases+sense+microbial+DNA+in+human+plasmacytoid+dendritic+cells.">Aspartate-glutamate-alanine-histidine box motif (DEAH)/RNA helicase A helicases sense microbial DNA in human plasmacytoid dendritic cells.</a> Proc Natl Acad Sci U S A. 107 (34), 15181-15186 (2010).</li><li>Booy, E. P., McRae, E. K., McKenna, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+characterization+of+G4+quadruplex+telomerase+RNA+unwinding+by+the+RNA+helicase+RHAU.">Biochemical characterization of G4 quadruplex telomerase RNA unwinding by the RNA helicase RHAU.</a> Methods Mol Biol. 1259, 125-135 (2015).</li><li>Lattmann, S., Giri, B., Vaughn, J. P., Akman, S. A., Nagamine, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+amino+terminal+RHAU-specific+motif+in+the+recognition+and+resolution+of+guanine+quadruplex-RNA+by+the+DEAH-box+RNA+helicase+RHAU.">Role of the amino terminal RHAU-specific motif in the recognition and resolution of guanine quadruplex-RNA by the DEAH-box RNA helicase RHAU.</a> Nucleic Acids Res. 38 (18), 6219-6233 (2010).</li></ol>

Denne protokollen er en svært effektiv ekspresjon, rensing og kvantifisering ordning for isolering av DHX36 genproduktet, G4-Resolvase1 (G4R1, også kalt RHAU og DHX36) (figur 1). Denne protokollen bruker to rensetrinn: His-tag affinitet rensing på kobolt affinitet perler og enzymatisk rensing på G4-DNA-konjugert perler. Sistnevnte steg er unik ved at den utnytter den stramme affinitet, høy spesifisitet, og katalytisk avkobling aktivitet som rG4R1 har for G4 strukturer. Den stramme affinitet for G4 ...

G4 strukturer er meget stabile nukleinsyre sekundære strukturer som dannes i løpet av guanin-rike regioner av DNA og RNA. G4 strukturer er stabilisert via Hoogsteen-binding interaksjoner og koordinere bonding innenfor den sentrale hulrom med enverdige kationer (ie. K + og Na +) som i vesentlig grad bidrar til den bemerkelsesverdige termiske stabilitet G4 strukturer 1, 2. Tidlige bioinformatikk studier antydet at det menneskelige genom inneholder&gt; 375.000 &quot;potensielle G4 dannende motiver&quot; 3, 4. Nyere studier beregninger tyder på at antallet av G4 motivene er høyere med en faktor på 2-5 5, mens en annen studie anslår 716,310 forskjellige potensielle G4 dannende sekvenser i det humane genom 6. G4-forming sekvenser er evolusjonært konserverte og ikke tilfeldig spredt igenomet. G4 motivene er beriket i genet som koder regioner, og i overkant av 40% av alle gener arrangører inneholde G4 motiver 7. Interessant nok har graden av anrikning av G4 motiver i et gen som er vist å foreslå funksjon av genet. For eksempel, proto-onkogener og gener som er involvert i utvikling har betydelig større berikelse av G4 strukturer enn tumorsuppressorgener 8, 9. Med høye termiske stabiliteten, en nesten allestedsnærværende tilstedeværelse i hele genomet, og potensial til å påvirke viktige cellulære prosesser, er det overraskende å finne at cellen har utviklet enzymer for å administrere disse strukturene. Et slikt enzym er G4 Resolvase1 (G4R1; også kalt RHAU og DHX36), som vi har betegnet som kilde for de fleste tetramolecular G4-DNA løse aktivitet i humane (HeLa celler) 10. Siden da har det blitt vist that G4R1 tett binder og katalytisk unwinds tetramolecular og unimolecular G4-DNA og G4-RNA med de trangeste rapporterte KDS for en G4-bindende protein 11, 12, 13. I tillegg har G4-oppløsnings aktivitet av G4R1 blitt implisert i en rekke biokjemiske og cellulære prosesser, herunder telomere / telomerase biologi 11, 14, 15, 16, transkripsjon og skjøting 17, 18, 19, 20, utviklings 21, hematopoese 21, og immunregulering 22, 23. Med en overvekt av G4 sekvenser spesifikt ligger hele genomet og diverse celle processes at G4R1 har nylig vært innblandet å være involvert med, evnen til å uttrykke og effektivt rense svært aktiv rG4R1 vil være av største betydning for å belyse de biokjemiske mekanismer og atferd av dette proteinet. Her viser vi en roman uttrykk og rensing ordningen (figur 1) som dro fordel av ATP-avhengige, G4-løse aktivitet rG4R1 å effektivt isolere aktivt enzym. Denne ordningen kan være innrettet til å rense andre ATP-avhengige nukleinsyre-enzymer som produktet av den enzymatiske reaksjon er ikke lenger et substrat for binding, slik tilfellet er for G4R1.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>TriEx4-DHX36 plasmid</td>
			<td>Addgene</td>
			<td>68368</td>
			<td></td>
		</tr>
		<tr>
			<td>Rosetta2(DE3)plysS competent cells</td>
			<td>Novagen</td>
			<td>71403-4</td>
			<td></td>
		</tr>
		<tr>
			<td>S.O.C medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>15544034&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco Terrific Broth</td>
			<td>Becton Dickinson</td>
			<td>243820</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Sigma-Aldrich</td>
			<td>C1919</td>
			<td>35 &#181;g/ml in bacterial plates/large cultures</td>
		</tr>
		<tr>
			<td>Carbenicillin (plant cell culture tested)</td>
			<td>Sigma-Aldrich</td>
			<td>C3416</td>
			<td>50 &#181;g/ml in bacterial plates/large cultures</td>
		</tr>
		<tr>
			<td>Isopropyl &#946;-D-1-thiogalactopyranoside (IPTG)</td>
			<td>Sigma-Aldrich</td>
			<td>I6758</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme (from chicken egg white)</td>
			<td>Sigma-Aldrich</td>
			<td>L6876</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M Tris-HCl pH=8</td>
			<td>Universal Scientific Supply Co.&#160;</td>
			<td>1963-B</td>
			<td>or From standard source</td>
		</tr>
		<tr>
			<td>1 M Tris-HCl pH=7</td>
			<td>Universal Scientific Supply Co.&#160;</td>
			<td>1966</td>
			<td>or From standard source</td>
		</tr>
		<tr>
			<td>1.5 M Tris-HCl, pH=8.8</td>
			<td></td>
			<td></td>
			<td>For casting resolving gel (for protein quantitation gel); From standard source</td>
		</tr>
		<tr>
			<td>1 M Tris-HCl, pH=6.8</td>
			<td></td>
			<td></td>
			<td>For casting stacking gel (for protein quantitation gel); From standard source</td>
		</tr>
		<tr>
			<td>1 M Tris-Acetate, pH=7.8</td>
			<td>Universal Scientific Supply Co.&#160;</td>
			<td>1981</td>
			<td>or From standard source</td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td></td>
			<td></td>
			<td>From standard source</td>
		</tr>
		<tr>
			<td>Magnesium chloride (1 M solution)</td>
			<td>Life Technologies</td>
			<td>AM9530G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Sigma-Aldrich</td>
			<td>S8750</td>
			<td></td>
		</tr>
		<tr>
			<td>20x SSC</td>
			<td>Universal Scientific Supply Co.&#160;</td>
			<td>1665</td>
			<td>or From standard source</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol (2-BME)</td>
			<td>Sigma-Aldrich</td>
			<td>63689</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>P8849</td>
			<td></td>
		</tr>
		<tr>
			<td>Leupeptin hemisulfate</td>
			<td>Sigma-Aldrich</td>
			<td>L8511</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin paramagnetic beads</td>
			<td>Promega</td>
			<td>Z5482</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 M EDTA, pH=8&#160;</td>
			<td>Universal Scientific Supply Co.&#160;</td>
			<td>0718</td>
			<td>or From standard source</td>
		</tr>
		<tr>
			<td>0.2 M EDTA, pH=6</td>
			<td>Universal Scientific Supply Co.&#160;</td>
			<td></td>
			<td>From standard source; initially adjust pH with NaOH, then adjust pH back down with HCl.&#160;&#160;</td>
		</tr>
		<tr>
			<td>A-lactalbumin (Type 1 from bovine milk)</td>
			<td>Sigma-Aldrich</td>
			<td>L5385</td>
			<td></td>
		</tr>
		<tr>
			<td>Cobalt metal affinity beads</td>
			<td>Clonetech</td>
			<td>635502</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Histidine</td>
			<td>Sigma-Aldrich</td>
			<td>H8000</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid, glacial</td>
			<td>Fisher Scientific</td>
			<td>A38-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 5&#39;-Triphosphate (from bacterial source)</td>
			<td>Sigma</td>
			<td>A7699</td>
			<td></td>
		</tr>
		<tr>
			<td>40% acrylamide/Bis solution (37.5:1)</td>
			<td>Biorad</td>
			<td>161-0148</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>50046</td>
			<td>to make protein gel running buffer (192 mM glycine, 25 mM Tris Base, 0.1% SDS)</td>
		</tr>
		<tr>
			<td>10 % Sodium dodecyl sulfate&#160;</td>
			<td>Universal Scientific Supply Co.&#160;</td>
			<td>1667</td>
			<td>to make protein gel running buffer (192 mM glycine, 25 mM Tris Base, 0.1% SDS); or From standard source</td>
		</tr>
		<tr>
			<td>10x TBE&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>11666703001</td>
			<td>or From standard source</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Fisher Scientific</td>
			<td>BP152-1</td>
			<td>to make protein gel running buffer (192 mM glycine, 25 mM Tris Base, 0.1% SDS); From standard source</td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Sigma-Aldrich</td>
			<td>411019</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>Sigma-Aldrich</td>
			<td>A3678</td>
			<td></td>
		</tr>
		<tr>
			<td>Broad Range Protein MW markers</td>
			<td>Promega</td>
			<td>V8491</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated Z33 oligo (&#34;Z33-Bio&#34;)</td>
			<td>Oligos Etc</td>
			<td></td>
			<td>5&#8217; AAA GTG ATG GTG GTG GGG GAA GGA TTC GGA CCT-biotin 3&#8217;</td>
		</tr>
		<tr>
			<td>TAMRA-Z33 oligo (&#34;Z33-TAM&#34;)</td>
			<td>Oligos Etc</td>
			<td></td>
			<td>5&#8217; TAMRA-AAA GTG ATG GTG GTG GGG GAA GGA TTC GGA CCT 3&#8217;</td>
		</tr>
		<tr>
			<td>Fluor-coated TLC plate</td>
			<td>Life Technologies</td>
			<td>AM10110</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll</td>
			<td>Sigma-Aldrich</td>
			<td>F2637</td>
			<td>30% in H2O</td>
		</tr>
		<tr>
			<td>Coomassie R-250</td>
			<td>Sigma-Aldrich</td>
			<td>27816</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A412</td>
			<td></td>
		</tr>
		<tr>
			<td>Multiband UV lamp</td>
			<td></td>
			<td></td>
			<td>Capable of emitting UV light at 365 nm</td>
		</tr>
		<tr>
			<td>Table-top centrifuge (with swinging bucket rotor)</td>
			<td></td>
			<td></td>
			<td>Capable of being cooled to 4 &#176;C</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td></td>
			<td></td>
			<td>Capable of being cooled to 4 &#176;C</td>
		</tr>
		<tr>
			<td>Digital Sonfier</td>
			<td>Branson</td>
			<td></td>
			<td>Or equivalent capable of delivering sonication pulses (30% amplitude, 2s ON 2s OFF)</td>
		</tr>
		<tr>
			<td>50 &#176;C water bath</td>
			<td></td>
			<td></td>
			<td>For formation of Z33 into quadruplex</td>
		</tr>
		<tr>
			<td>37 &#176;C incubator for bacteria</td>
			<td></td>
			<td></td>
			<td>For bacterial transformations and initial overnight growth of large cultures of Rosetta2 E. coli transformed with TriEx4-DHX36</td>
		</tr>
		<tr>
			<td>37 &#176;C/14&#160; &#176;C shaking incubator for bacteria</td>
			<td></td>
			<td></td>
			<td>For growth and protein induction of large cultures of Rosetta2 E. coli transformed with TriEx4-DHX36</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td></td>
			<td></td>
			<td>capable of reading OD600; capable of reading oligomer concentrations based on base sequence (such as Biorad SmartSpec 3000)</td>
		</tr>
		<tr>
			<td>Thermometer</td>
			<td></td>
			<td></td>
			<td>From standard source</td>
		</tr>
		<tr>
			<td>PCR strip tubes</td>
			<td></td>
			<td></td>
			<td>From standard source</td>
		</tr>
		<tr>
			<td>15- and 50-ml centrifuge tubes (polypropylene)</td>
			<td></td>
			<td></td>
			<td>From standard source</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes (2.0 ml)</td>
			<td></td>
			<td></td>
			<td>From standard source</td>
		</tr>
		<tr>
			<td>500 ml centrifuge bottles (polypropylene)</td>
			<td>Thermo Scientific</td>
			<td>3141-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard array of pipet tips and serological pipettes</td>
			<td></td>
			<td></td>
			<td>From standard source</td>
		</tr>
		<tr>
			<td>Gel-loading tips</td>
			<td></td>
			<td></td>
			<td>From standard source</td>
		</tr>
		<tr>
			<td>Automatic repeating pipette</td>
			<td></td>
			<td></td>
			<td>For quick aliquoting of rG4R1; From standard source</td>
		</tr>
		<tr>
			<td>Thermal cycler</td>
			<td></td>
			<td></td>
			<td>From standard source</td>
		</tr>
		<tr>
			<td>Liquid Nitrogen</td>
			<td></td>
			<td></td>
			<td>From standard source</td>
		</tr>
		<tr>
			<td>Dry ice</td>
			<td></td>
			<td></td>
			<td>From standard source</td>
		</tr>
		<tr>
			<td>Laemlli sample buffer&#160;</td>
			<td>Biorad</td>
			<td>161-0737</td>
			<td></td>
		</tr>
		<tr>
			<td>Apparatus for running large slab gels</td>
			<td>Biorad</td>
			<td></td>
			<td>We have used the Protean II xi cell apparatus from Biorad</td>
		</tr>
		<tr>
			<td>Magnet</td>
			<td>Life Technologies</td>
			<td>12301D</td>
			<td>We use a magnet from One Lambda (Now a Thermo Fisher Scientific brand); and Life is also a subsidiary of Thermo, and thus the magnet listed here should be a suitable replacement</td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td></td>
			<td></td>
			<td>From standard source</td>
		</tr>
		<tr>
			<td>Filter paper and funnel</td>
			<td></td>
			<td></td>
			<td>From standard source</td>
		</tr>
		<tr>
			<td>Glass casserole dish</td>
			<td></td>
			<td></td>
			<td>From standard source</td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td></td>
			<td></td>
			<td>From standard source</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td></td>
			<td></td>
			<td>From standard source</td>
		</tr>
		<tr>
			<td>Clear sheet protectors</td>
			<td></td>
			<td></td>
			<td>From standard source</td>
		</tr>
		<tr>
			<td>Scanner and associated TWAIN software</td>
			<td></td>
			<td></td>
			<td>From standard source</td>
		</tr>
		<tr>
			<td>Image analysis software</td>
			<td></td>
			<td></td>
			<td>Such as Fuji Multiguage, or equivalent</td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td></td>
			<td></td>
			<td>Or equivalent&#160;</td>
		</tr>
	</tbody>
</table>

a g-quadruplex dna-affinity approach for purification of enzymatically active g4 resolvase1

Høyere ordens nukleinsyre strukturer kalles G-quadruplexes (G4S G4 strukturer) kan dannes i guanin rike regioner i både DNA og RNA og er svært termisk stabilt. Det er&gt; 375.000 putative G4 dannende sekvenser i det humane genom, og de er anriket i promotorområdene, ikke-translaterte regioner (UTR), og inne i telomeric gjenta. På grunn av potensialet for disse strukturene for å påvirke cellulære prosesser, som for eksempel replikasjon og transkripsjon, har cellen utviklet enzymer for å håndtere dem. Et slikt enzym er G4 Resolvase 1 (G4R1), som ble biokjemisk co karakterisert ved vårt laboratorium og Nagamine et al. og funnet å binde ekstremt tett til både G4-DNA og G4-RNA (K d i det lav-pM området). G4R1 er kilden for de fleste av G4-løse-aktivitet i HeLa-cellelysatene og har siden blitt implisert å spille en rolle i metabolismen telomere, lymfe utvikling, gentranskripsjon, hematopoese, og immunovervåkning. Evnen til å efstrekkelig uttrykke og rense katalytisk aktive G4R1 er av betydning for laboratorier som er interessert i å få mer innsikt i den kinetiske samspillet mellom G4 strukturer og G4-løse enzymer. Her beskriver vi en detaljert fremgangsmåte for rensing av rekombinant G4R1 (rG4R1). Den beskrevne fremgangsmåten omfatter den tradisjonelle affinitets-baserte rensing av en C-terminal histidin-merket enzym uttrykt i humane kodonoptimalisert bakterier med utnyttelse av evnen til å binde rG4R1 og koble G4-DNA for å rense meget aktivt enzym i en ATP- avhengig eluering trinn. Protokollen omfatter også en kvalitetskontrolltrinn hvor den enzymatiske aktiviteten til rG4R1 blir målt ved å undersøke evnen til renset enzym for å koble av G4-DNA. En fremgangsmåte er også beskrevet som gjør det mulig for kvantifisering av renset rG4R1. Alternative tilpasninger av denne protokollen blir diskutert.

Denne protokollen (figur 1) gir rutinemessig nesten ren, katalytisk aktive rG4R1. Som et mål på enzymatisk aktivitet, blir det vanligvis observert at 50% av 0,2 pmol av TAMRA-merket tetramolecular G4-DNA blir omdannet til monomerer innenfor 0,2 til 0,013 mL rekke rG4R1, som analysert ved hjelp av G4 aktivitetsanalyse som er beskrevet ovenfor (figur 2). Coomassie-farging av renset rG4R1 angir et enkelt bånd ved de forventede 120 kDa størrelse, med en mindre forurensende bånd ved ~ 7...

Watch this Scientific Journal Video about A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1 at JoVE.com

A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1

G4 Resolvase1 binder seg til G-quadruplex (G4) strukturer med de trangeste rapporterte affinitet for en G4-bindende protein og representerer flertallet av G4-DNA slappe aktivitet i HeLa celler. Vi beskriver en roman protokoll som utnytter affinitet og ATP-avhengig avvikling aktiviteten til G4-Resolvase1 å spesifikt rense katalytisk aktive rekombinant G4R1.

En G-quadruplex DNA-affinitet tilnærming for rensing av enzymatisk aktivt G4 Resolvase1

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly ...

Research

JoVE Journal

Biochemistry

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

Regissert Protein Emballasje innen ytre membran Blemmer fra<em&gt; Escherichia coli</em&gt;: Design, produksjon og rensing

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique ...

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

En Facile og effektiv tilnærming for produksjon av Reversibel disulfide tverrbundet Miceller

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

Expression, Purification, and Antimicrobial Activity of S100A12

Ekspresjon, rensing og antimikrobiell aktivitet av 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 ...

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Metode for effektiv Refolding og rensing av Chemoreceptor Ligand bindende domene

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Rensing og rekonstituering av TRPV1 for Spectroscopic analyse

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

Anaerob Protein rensing og kinetisk analyse via oksygen elektrode for å studere DesB Dioxygenase aktivitet og hemming

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic ...

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

Identifikasjon av romanen CK2 Kinase underlag med en allsidig biokjemiske tilnærming

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets ...

A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products

En tilpasningsvennlig tilnærming for enzymatisk produksjon og rensing av Diterpenoid naturlige produkter

Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Enkeltcelleanalyse av transkripsjonsaktive alleler av enkeltmolekyl fisk

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

Cellefraksjonering av U937-celler ved isopycnic tetthetsgradientrensing

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and ...