Name: a g-quadruplex dna-affinity approach for purification of enzymatically active g4 resolvase1
Uploaded: 2017-03-18T14:00:00
Description: Watch this Scientific Journal Video about A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1 at JoVE.com

We would like to thank our funding sources, including a generous gift from the Ware Foundation (to J.P.V.), The National Institutes of HealthGrant T32-CA079448 (to P.J.S.), and Ball State University startup funds (to P. J. S.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

1. Preparation of G4-DNA Structures to be Used for the Purification of rG4R1 (Formation of Biotinylated G4-DNA G-Quadruplex)
<ol>
	<li>Order the following DNA oligomer, called Z33-Bio, at a 1 &#181;mole-scale: 5&#8217; AAA GTG ATG GTG GTG GGG GAA GGA TTC GGA CCT-biotin 3&#8217;. Ensure that the biotin moiety is at the 3&#8217; end of the oligomer.</li>
	<li>Prepare 10x G4 buffer: 450 mM Tris-HCl pH 8, 25 mM EDTA, and 2,500 mM NaCl.</li>
	<li>Resuspend the Z33 oligomer in 250 &#181;L of water (so that the oligomer conce...

<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>

This protocol represents a highly efficient expression, purification, and quantification scheme for the isolation of the DHX36 gene product, G4-Resolvase1 (G4R1, also called RHAU and DHX36) (Figure 1). This protocol utilizes two purification steps: His-tag affinity purification on cobalt affinity beads and enzymatic purification on G4-DNA-conjugated beads. The latter step is unique in that it takes advantage of the tight affinity, high specificity, and catalytic unwinding activity that rG4R1 has for G4 s...

G4 structures are highly stable nucleic acid secondary structures that form within guanine-rich regions of DNA and RNA. G4 structures are stabilized via Hoogsteen-bonding interactions and coordinate bonding within the central cavity with monovalent cations (i.e.&#160;K+ and Na+) that significantly contribute to the remarkable thermal stability of G4 structures1,2. Early bioinformatics studies suggested that the human genome contains &#62;375,000 &#8220;potential G4-forming motifs&#8221;3,4. More recent study estimates suggest that the number of G4 motifs is higher by a factor of 2-55, while another study predicts 716,310 distinct potential G4-forming sequences in the human genome6. G4-forming sequences are evolutionarily conserved and not randomly dispersed in the genome. G4 motifs are enriched in gene coding regions, and upwards of 40% of all gene promoters contain G4 motifs7. Interestingly, the degree of enrichment of G4 motifs in a gene has been demonstrated to suggest the function of the gene. For example, proto-oncogenes and genes involved in development have significantly greater enrichment of G4 structures than tumor suppressor genes8,9.

With high thermal stabilities, a nearly ubiquitous presence throughout the genome, and the potential to significantly affect major cellular processes, it is unsurprising to find that the cell has evolved enzymes to manage these structures. One such enzyme is G4 Resolvase1 (G4R1; also called RHAU and DHX36), which we characterized as the source of the majority of tetramolecular G4-DNA resolving activity in human (HeLa) cells10. Since then, it has been shown that G4R1 tightly binds and catalytically unwinds tetramolecular and unimolecular G4-DNA and G4-RNA with the tightest reported KDs for a G4-binding protein11,12,13. Additionally, the G4-resolving activity of G4R1 has been implicated in a wide range of biochemical and cellular processes, including telomere/telomerase biology11,14,15,16, transcription and splicing17,18,19,20, development21, hematopoiesis21, and immune regulation22,23. With a preponderance of G4 sequences specifically situated throughout the genome and the diverse cellular processes that G4R1 has recently been implicated to be involved with, the ability to express and efficiently purify highly active rG4R1 will be of the utmost importance for elucidating the biochemical mechanisms and behaviors of this protein.

Here, we demonstrate a novel expression and purification scheme (Figure 1) that takes advantage of the ATP-dependent, G4-resolving activity of rG4R1 to efficiently isolate active enzyme. This scheme could be adapted to purify other ATP-dependent nucleic acid enzymes for which the product of the enzymatic reaction is no longer a substrate for binding, as is the case 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

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 thermally stable. There are &#62;375,000 putative G4-forming sequences in the human genome, and they are enriched in promoter regions, untranslated regions (UTRs), and within the telomeric repeat. Due to the potential for these structures to affect cellular processes, such as replication and transcription, the cell has evolved enzymes to manage them. One such enzyme is G4 Resolvase 1 (G4R1), which was biochemically co-characterized by our laboratory and Nagamine et al. and found to bind extremely tightly to both G4-DNA and G4-RNA (Kd in the low-pM range). G4R1 is the source of the majority of G4-resolving activity in HeLa cell lysates and has since been implicated to play a role in telomere metabolism, lymph development, gene transcription, hematopoiesis, and immune surveillance. The ability to efficiently express and purify catalytically active G4R1 is of importance for laboratories interested in gaining further insight into the kinetic interaction of G4 structures and G4-resolving enzymes. Here, we describe a detailed method for the purification of recombinant G4R1 (rG4R1). The described procedure incorporates the traditional affinity-based purification of a C-terminal histidine-tagged enzyme expressed in human codon-optimized bacteria with the utilization of the ability of rG4R1 to bind and unwind G4-DNA to purify highly active enzyme in an ATP-dependent elution step. The protocol also includes a quality-control step where the enzymatic activity of rG4R1 is measured by examining the ability of the purified enzyme to unwind G4-DNA. A method is also described that allows for the quantification of purified rG4R1. Alternative adaptations of this protocol are discussed.

This protocol (Figure 1) routinely yields nearly pure, catalytically active rG4R1. As a measure of enzymatic activity, it is typically observed that 50% of 0.2 pmol of TAMRA-labeled tetramolecular G4-DNA is converted into monomers within the 0.2 - 0.013 &#181;L range of rG4R1, as assayed by the G4 activity assay outlined above (Figure 2). Coomassie staining of purified rG4R1 indicates a single band at the expected 120 kDa size, with a minor contaminating band at ~75 kDa, which may repres...

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 binds to G-quadruplex (G4) structures with the tightest reported affinity for a G4-binding protein and represents the majority of the G4-DNA unwinding activity in HeLa cells. We describe a novel protocol that harnesses the affinity and ATP-dependent unwinding activity of G4-Resolvase1 to specifically purify catalytically active recombinant G4R1.

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

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

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

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

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

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

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

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

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

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

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