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 ...

The overall goal of this method is to isolate the G-quadruplex helicase G4 Resolvase1 protein from a bacterial expression system using a unique ATP dependent purification step to isolate nearly pure and catalytically active enzyme. This method can help researchers obtain nearly pure and catalytically active recombinant G4 Resolvase1 protein for us in a wide of in vitro biochemical assays. The main advantage of this technique is that it excludes misfolded or otherwise catalytically inactive enzyme from the protein preparation.The idea to use ATP to elute the G-quadruplex helicase originated after being unable to elute the catalytically active enzyme off the G4 beads, even at exceptionally high salt concentrations. Prepare and grow large bacterial cultures expressing G-quadruplex Resolvase1 or G4R1 as described in the text protocol. Thaw the bacterial pellet in three milliliters of TN buffer at room temperature.Hold the bottles in hand and swirl to thaw and resuspend the bacterial pellet. Once thawed and relatively evenly suspended bring the volume up to five milliliters with TN buffer. Next, dissolve 20 milligrams of lysozyme in 250 microliters of water per culture and add to the resuspended bacteria.Allow the lysozyme to digest the bacteria for approximately five to 10 minutes while swirling the bottles in hand. The color of the suspension will begin to lighten slightly as the digestion proceeds, and the suspension will be relatively non viscous. Add 250 microliters of protease inhibitor cocktail abbreviated PIC.Then, add one 10 microliter aliquot of leupeptin and mix thoroughly. Place the suspension on ice and add 10 milliliters of cold TN buffer and 22 microliters of beta mercaptoethanol or BME. Transfer the suspension to a 50 milliliter tube.Sonicate the bacteria on ice with a digital sonicator set to 30%amplitude. Pulse at two seconds on and two seconds off for one minute. Repeat the sonication three times, allowing the samples to cool on ice for at least two minutes between sonication steps.Next, add an equal volume of cold four X saline sodium citrate or SSC plus BME, PIC, and leupeptin and mix the suspension. In a tabletop centrifuge pre cooled to four degrees Celsius centrifuge the lysates at 2, 300 times G for 20 minutes. Then transfer the supernatant to fresh pre cooled 50 milliliter tubes.Take one milliliter of streptavidin paramagnetic beads, or SPB suspension per culture, and transfer it to a 1.5 milliliter microcentrifuge tube. Pellet the SPB with a magnet. Then wash the SPB twice with two X SSC plus five millimolar EDTA PH8.Next, resuspend the washed SPB in 200 microliters of the same solution. Add one three OD aliquot of G4 DNA to the SPB suspension. Mix quickly by pipetting up and down several times.Then, place the tubes on a rotator and rotate at room temperature for at least 30 minutes. After this period of rotation, block the G4 bound SPB by adding one milliliter of 0.4%lactalbumin, and keep on ice until needed. To bind histidine tagged recombinant G4R1 to cobalt beads add one milliliter of cobalt bead slurry to each 50 milliliter tube containing the clarified bacterial lysate.Incubate the mixture for 20 minutes at room temperature on a rotator. Following incubation spin down cobalt beads at 110 times G for five minutes in a four degrees Celsius tabletop centrifuge. Aspirate the liquid carefully, and leave a couple milliliters of liquid so as not to disturb the pelleted cobalt beads.Then, wash the beads with 10 to 15 milliliters of cold four X SSC plus BME, and pellet the beads as before. Pour the next clarified lysate onto the pelleted cobalt beads. Incubate for a further 20 minutes at room temperature on the rotator, and wash the beads with 10 to 15 milliliters of four X SSC plus BME.Then, pellet the beads at 100 times G for five minutes at four degrees Celsius. After a second wash, aspirate the liquid and leave about two milliliters of beads and liquid at the bottom of the tube. Transfer the protein bound cobalt beads to a pre cooled two milliliter tube by using a one milliliter wide bore pipette tip.Briefly centrifuge the beads in the two milliliter tube at high speed in a microcentrifuge at four degrees Celsius. Gently remove and discard the supernatant by pipetting being careful not to lose the protein bound cobalt beads. Pipette 0.5 milliliters of histidine elution buffer, or HEB onto the beads and resuspend them by inverting the tube.Then incubate the cobalt beads in HEB on a rotator for five minutes in a cold room. Next, centrifuge the suspension at 18, 000 times G in a four degrees Celsius microcentrifuge for one minute. Gently remove the protein containing supernatant by careful pipetting leaving a small amount of liquid on top of the cobalt beads.Transfer the supernatant to a pre cooled 15 milliliter tube. Repeat these steps for a total of three HEB elutions. Then elute once with 0.2 molar EDTA at PH 6.0.The color of the cobalt beads will change from pink to white as the EDTA chelates the cobalt. After this fourth and final elution has been transferred to the 15 milliliter tube, pipette the residual elution buffer from the cobalt beads by using a gel loading tip on the end of a one milliliter pipette tip. By plunging the tip to the bottom of the tube residual protein containing elution buffer can be collected.Pilot the prepared G4 SPB to the side of the tube with a magnet and discard the supernatant. Then, add one milliliter of three X res buffer to the G4 SPB and pipette to mix. Pipette the G4 SPB suspended in three X res buffer into the 15 milliliter tube containing approximately two milliliters of protein containing HEB EDTA.Incubate for 15 minutes in a 37 degree Celsius water bath with occasional agitation so the beads do not settle. After washing the protein bound G4 SPB as described in the text protocol pellet the G4 SPB with a magnet on ice and resuspend the beads in 100 microliters of pre warmed elution buffer. Immediately transfer the beads to a pre warmed PCR tube and incubate for 30 seconds at 37 degrees Celsius.Promptly add 12 microliters of five molar sodium chloride and pipette up and down vigorously 20 to 30 times. The combination of the high salt and ATP contained in the elution buffer serves to elute recombinant G4R1 from the G4 beads. Immediately pellet the G4 SPB with a magnet, and transfer the protein containing elute to a fresh PCR tube on ice.After repeating the elution and transfer the end product is approximately 200 microliters of elution buffer containing purified recombinant G4R1. Finally, perform the quality control enzymatic activity assay to test whether highly active recombinant G4R1 has been purified as described in the text protocol. The G4 assay provides a measure of recombinant G4R1 enzymatic activity.Within the 0.2 to 0.013 microliter range in the enzyme it is typically observed that 50%of 0.2 picomoles of tetramethylrhodamine labeled tetramolecular G4 DNA is converted into monomers. Coomassie standing of purified recombinant G4R1 indicates a single band at the expected 120 kilodalton size with a minor contaminating band at approximately 75 kilodaltons. This band may represent truncated recombinant G4R1 that has maintained is ability to bind G4 DNA beads into elute in an ATP dependent manner.The band of interest is quantified against a protein standard curve. This protocol typically obtains 200 microliters of 20 to 100 nanomolar purified enzyme per one liter of bacteria culture. After watching this video, you should have a good understanding of how to purify G4 Resolvase1 protein from a bacterial expression system using a unique ATP dependent purification step to isolate nearly pure and catalytically active enzyme.Once mastered, and with prior preparation of the frozen bacteria pellets, the purification of recombinant G4R1 can be performed in approximately six hours. To maintain the optimal catalytic activity of the enzyme it's important to keep the preparation on ice, unless otherwise noted. Always use protease inhibitors.Avoid bubble formation in the tubes. Freeze purified enzyme quickly, and avoid unnecessary freeze thaw cycles. After its development, this technique paved the way for researchers in the field to accurately define the binding affinities and helicase activities of this protein to a wide range of DNA and RNA substrates.This method can also be adapted for the purification of other ATP dependent nucleic acid enzymes for which the product of the enzymatic reaction is no longer a binding substrate for the enzyme.

a-g-quadruplex-dna-affinity-approach-for-purification-enzymatically

Research

JoVE Journal

Biochemistry

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 applications for OMV where traditional nanoparticles are proving too difficult to synthesize. To date, all Gram-negative bacteria have been shown to produce OMV demonstrating packaging of a variety of cargo that includes small molecules, peptides, proteins and genetic material. Based on their diverse cargo, OMV are implicated in many biological processes ranging from cell-cell communication to gene transfer and delivery of virulence factors depending upon which bacteria are producing the OMV. Only recently have bacterial OMV become accessible for use across a wide range of applications through the development of techniques to control and direct packaging of recombinant proteins into OMV. This protocol describes a method for the production, purification, and use of enzyme packaged OMV providing for improved overall production of recombinant enzyme, increased vesiculation, and enhanced enzyme stability. Successful utilization of this protocol will result in the creation of a bacterial strain that simultaneously produces a recombinant protein and directs it for OMV encapsulation through creating a synthetic linkage between the recombinant protein and an outer membrane anchor protein. This protocol also details methods for isolating OMV from bacterial cultures as well as proper handling techniques and things to consider when adapting this protocol for use for other unique applications such as: pharmaceutical drug delivery, medical diagnostics, and environmental remediation.

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

A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented.

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. Improving drug delivery to maximize therapeutic outcomes and to reduce drug-associated side effects are some of the cornerstones of present-day nanomedicine. Nanoparticles in particular have found a wide application in cancer treatment. Nanoparticles that offer a high degree of flexibility in design, application, and production based on the tumor microenvironment are projected to be more effective with rapid translation into clinical practice. The polymeric micellar nano-carrier is a popular choice for drug delivery applications.
In this article, we describe a simple and effective protocol for synthesizing drug-loaded, disulfide cross-linked micelles based on the self-assembly of a well-defined amphiphilic linear-dendritic copolymer (telodendrimer, TD). TD is composed of polyethylene glycol (PEG) as the hydrophilic segment and a thiolated cholic acid cluster as the core-forming hydrophobic moiety attached stepwise to an amine-terminated PEG using solution-based peptide chemistry. Chemotherapy drugs, such as paclitaxel (PTX), can be loaded using a standard solvent evaporation method. The O2-mediated oxidation was previously utilized to form intra-micellar disulfide cross-links from free thiol groups on the TDs. However, the reaction was slow and not feasible for large-scale production. Recently, an H2O2-mediated oxidation method was explored as a more feasible and efficient approach, and it was 96 times faster than the previously reported method. Using this approach, 50 g of PTX-loaded, disulfide cross-linked nanoparticles have been successfully produced with narrow particle size distribution and high drug loading efficiency. The stability of the resulting micelle solution is analyzed using disrupting conditions such as co-incubation with a detergent, sodium dodecyl sulfate, with or without a reducing agent. The drug-loaded, disulfide cross-linked micelles demonstrated less hemolytic activity when compared to their non-cross-linked counterparts.

To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

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 S100 proteins have the capacity to bind transition metals with high affinity and effectively sequester them away from invading microbial pathogens in a process that is termed "nutritional immunity". S100A12 (EN-RAGE) binds both zinc and copper and is highly abundant in innate immune cells such as macrophages and neutrophils. We report a refined method for the expression, enrichment and purification of S100A12 in its active, metal-binding configuration. Utilization of this protein in bacterial growth and viability analyses reveals that S100A12 has antimicrobial activity against the bacterial pathogen, Helicobacter pylori. The antimicrobial activity is predicated on the zinc-binding activity of S100A12, which chelates nutrient zinc, thereby starving H. pylori which requires zinc for growth and proliferation.

Here, we present a method to express and purify S100A12 (calgranulin C). We describe a protocol to measure its antimicrobial activity against the human pathogen H. pylori.

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 greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

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 and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.

This article describes specific methods to obtain biochemical quantities of detergent-solubilized TRPV1 for spectroscopic analysis. The combined protocols provide biochemical and biophysical tools that can be adapted to facilitate structural and functional studies for mammalian ion channels in a membrane-controlled environment.

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 purification methods. This manuscript illustrates the technical details involved in the anaerobic purification process, including the preparation of buffers and reagents, the methods for column chromatography in a glove box, and the desalting of the protein prior to kinetics. Also described are the methods for preparing and using an oxygen electrode to perform kinetic characterization of an oxygen-utilizing enzyme. These methods are illustrated using the dioxygenase enzyme DesB, a gallate dioxygenase from the bacterium Sphingobium sp. strain SYK-6.

Here we present a protocol for anaerobic protein purification, anaerobic protein concentration, and subsequent kinetic characterization using an oxygen electrode system. The method is illustrated using the enzyme DesB, a dioxygenase enzyme which is more stable and active when purified and stored in an anaerobic environment.

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

Expression and Purification of Nuclease-Free Oxygen Scavenger Protocatechuate 3,4-Dioxygenase

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, fluorophores are prone to loss of signal via photobleaching by dissolved oxygen (O2). To prevent photobleaching and extend the fluorophore lifetime, oxygen scavenging systems (OSS) are employed to reduce O2. Commercially available OSS may be contaminated by nucleases that damage or degrade nucleic acids, confounding interpretation of experimental results. Here we detail a protocol for the expression and purification of highly active Pseudomonas putida protocatechuate-3,4-dioxygenase (PCD) with no detectable nuclease contamination. PCD can efficiently remove reactive O2 species by conversion of the substrate protocatechuic acid (PCA) to 3-carboxy-cis,cis-muconic acid. This method can be used in any aqueous system where O2 plays a detrimental role in data acquisition. This method is effective in producing highly active, nuclease free PCD in comparison with commercially available PCD.

Protocatechuate 3,4-dioxygenase (PCD) can enzymatically remove free diatomic oxygen from an aqueous system using its substrate protocatechuic acid (PCA). This protocol describes the expression, purification, and activity analysis of this oxygen scavenging enzyme.

Single molecule (SM) microscopy is used in the study of dynamic molecular interactions of fluorophore labeled biomolecules in real time. However, ...

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 functions in developmental processes, interorganismal interactions, and environmental adaptation. Due to these various bioactivities, many diterpenoids are also of economic importance as pharmaceuticals, food additives, biofuels, and other bioproducts. Advanced genomics and biochemical approaches have enabled a rapid increase in the knowledge of diterpenoid-metabolic genes, enzymes, and pathways. However, the structural complexity of diterpenoids and the narrow taxonomic distribution of individual compounds in often only a single species remain constraining factors for their efficient production. Availability of a broader range of metabolic enzymes now provide resources for producing diterpenoids in sufficient titers and purity to facilitate a deeper investigation of this important metabolite group. Drawing on established tools for microbial and plant-based enzyme co-expression, we present an easily operated and customizable protocol for the enzymatic production of diterpenoids in either Escherichia coli or Nicotiana benthamiana, and the purification of desired products via silica chromatography and semi-preparative HPLC. Using the group of maize (Zea mays) dolabralexin diterpenoids as an example, we highlight how modular combinations of diterpene synthase (diTPS) and cytochrome P450 monooxygenase (P450) enzymes can be used to generate different diterpenoid scaffolds. Purified compounds can be used in various downstream applications, such as metabolite structural analyses, enzyme structure-function studies, and in vitro and in planta bioactivity experiments.

Here we present easy to use protocols for producing and purifying diterpenoid metabolites through the combinatorial expression of biosynthetic enzymes in Escherichia coli or Nicotiana benthamiana, followed by chromatographic product purification. The resulting metabolites are suitable for various studies including molecular structure characterization, enzyme functional studies, and bioactivity assays.

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 population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

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

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Caenorhabditis elegans has been an important model system for biological research since it was introduced in 1963. However, C. elegans has not been fully utilized in the biochemical study of biological reactions using its nuclear extracts such as in vitro transcription and DNA replication. A significant hurdle for using C. elegans in biochemical studies is disrupting the nematode&#39;s thick outer cuticle without sacrificing the activity of the nuclear extract. While several methods are used to break the cuticle, such as Dounce homogenization or sonication, they often lead to protein instability. There are no established protocols for isolating active nuclear proteins from larva or adult C. elegans for in vitro reactions. Here, the protocol describes in detail the homogenization of larval stage 4 C. elegans using a Balch homogenizer. The Balch homogenizer uses pressure to slowly force the animals through a narrow gap breaking the cuticle in the process. The uniform design and precise machining of the Balch homogenizer allow for consistent grinding of animals between experiments. Fractionating the homogenate obtained from the Balch homogenizer yields functionally active nuclear extract that can be used in an in vitro method for assaying transcription activity of C. elegans.

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Here, we describe a detailed protocol for isolating active nuclear extract from larval stage 4 C. elegans and visualizing transcription activity in an in vitro system.

Caenorhabditis elegans has been an important model system for biological research since it was introduced in 1963. However, C. elegans has not been fully ...