우리는 (JPV까지) 도자기 재단의 관대 한 선물을 포함하여, 우리의 재원을 감사드립니다 (PJS에) (PJS에) HealthGrant T32-CA079448 및 볼 주립 대학 시작 기금의 국립 연구소. 자금 제공자는 연구 설계, 자료 수집 및 분석, 게시하는 결정, 또는 원고의 준비에 아무런 역할을하지 않았다.

<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>

저자는 공개 아무것도 없어.

이 프로토콜은 DHX36 유전자 산물, G4-Resolvase1 (또한 RHAU 및 DHX36라고 G4R1) (그림 1)의 분리를위한 고효율 발현, 정제 및 정량 방식을 나타냅니다. G4-DNA 접합 구슬에 대한 그의 태그 친 화성 코발트 친 화성 구슬 정화 및 효소 정제 :이 프로토콜은 두 개의 정제 단계를 사용합니다. 후자 단계는 rG4R1는 G4 구조물 가진 꽉 선호도 높은 특이성 및 풀림 촉매 활성을 활용하는 것을 독특하다. G4 구?...

G4 구조는 DNA와 RNA의 구아닌이 풍부한 지역 내에 형성 안정성이 높은 핵산 차 구조이다. G4 구조 훅 스틴 - 결합 상호 작용을 통해 안정화 크게 G4 구조 (1, 2)의 주목할만한 열적 안정성에 기여하는 가의 양이온 (예. K +와 + 나)와 중앙 공동 내에 결합을 조정한다. 초기 생물 정보학 연구는 인간 게놈&gt; 375,000 &quot;잠재적 인 G4 형성 모티브&quot;3, 4 포함하는 것이 좋습니다. 최근의 연구 평가는 다른 연구는 인간 게놈 6 716310 별개의 잠재적 인 G4 형성 순서를 예측하면서 G4 주제의 수는 2-5 5 배 정도 높은 것이 좋습니다. G4는-형성하는 서열은 진화 적으로 보존 무작위로 분산되지 않습니다게놈. G4 주제는 유전자의 코딩 지역에서 농축되고, 모든 유전자 프로모터의 40 %의 위쪽으로는 G4 모티브 (7)를 포함한다. 흥미롭게도, 유전자의 G4 모티프의 농축 정도는 유전자의 기능을 제시하는 것으로 입증되었다. 예를 들면, 현상에 관여하는 프로토 - 암 유전자 및 유전자는 종양 억제 유전자 8,9보다 G4 구조 훨씬 더 농축있다. 높은 열 안정성으로 게놈 전체에서 거의 어디에나 존재 상당히 중요한 세포 과정에 영향을 미칠 가능성이, 상기 셀은 이러한 구조를 관리하기 위해 효소를 진화 것을 발견 놀랍지이다. 우리는 인간 (헬라) 세포 10 tetramolecular G4-DNA 해결 활동의 대부분의 소스로 특징으로 하나의 효소는 G4 Resolvase1 (또한 RHAU 및 DHX36라고 G4R1)입니다. 그 이후로,이 표시되어 그쪽으로t G4R1 단단히 바인딩 및 촉매 G4 결합 단백질 11, 12, 13에 대한 엄격한보고 된 KDS와 tetramolecular 및 단 분자 G4-DNA와 G4-RNA가 풀릴. 또한 G4R1의 G4 분해하는 활성 텔로미어 / 텔로 머라 생물학 11, 14, 15, 16, 전사 및 접합 17, 18, 19, 20, 개발 21 조혈 포함 생화학 및 세포 과정의 다양한 연루되었는지 (21), 및 면역 조절 22, 23. G4 시퀀스의 우세는 특히 게놈 및 다양한 휴대 페이지에 걸쳐 위치하고로G4R1 최근 표현 효율적이 단백질의 생화학 적 메커니즘과 동작을 밝히기 위해 rG4R1가 매우 중요 할 것이다 활성이 높은 정화 능력에 관여하는 연루되었는지 rocesses. 여기서 우리는 새로운 표현하고 효율적으로 활성 효소를 분리 rG4R1의 ATP 의존, G4-해결 활동을 활용 정화 방식 (그림 1)를 보여줍니다. G4R1 대한 경우와 같이,이 방식은, 효소 반응의 생성물은 더 이상 바인딩 기재되는 다른 ATP 의존성 핵산 효소를 정제하도록 채택 될 수있다.

<table><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&amp;nbsp;</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 &amp;micro;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 &amp;micro;g/mL in bacterial plates/large cultures</td></tr><tr><td>Isopropyl &amp;beta;-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.&amp;nbsp;</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.&amp;nbsp;</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.&amp;nbsp;</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&amp;nbsp;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.&amp;nbsp;</td><td>1665</td><td>or from standard source</td></tr><tr><td>&amp;beta;-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&amp;nbsp;</td><td>Universal Scientific Supply Co.&amp;nbsp;</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.&amp;nbsp;</td><td></td><td>From standard source; initially adjust pH with NaOH, then adjust pH back down with HCl.&amp;nbsp;&amp;nbsp;</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&#039;-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 (SDS)</td><td>Universal Scientific Supply Co.&amp;nbsp;</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&amp;nbsp;</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 (APS)</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 (&quot;Z33-Bio&quot;)</td><td>Oligos Etc</td><td></td><td>5&amp;rsquo; AAA GTG ATG GTG GTG GGG GAA GGA TTC GGA CCT-biotin 3&amp;rsquo;</td></tr><tr><td>TAMRA-Z33 oligo (&quot;Z33-TAM&quot;)</td><td>Oligos Etc</td><td></td><td>5&amp;rsquo; TAMRA-AAA GTG ATG GTG GTG GGG GAA GGA TTC GGA CCT 3&amp;rsquo;</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 H&lt;sub&gt;2&lt;/sub&gt;O</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 &amp;deg;C</td></tr><tr><td>Microcentrifuge</td><td></td><td></td><td>Capable of being cooled to 4 &amp;deg;C</td></tr><tr><td>Digital Sonfier</td><td>Branson</td><td></td><td>Or equivalent capable of delivering sonication pulses (30% amplitude, 2 s ON/2 s OFF)</td></tr><tr><td>50 &amp;deg;C water bath</td><td></td><td></td><td>For formation of Z33 into quadruplex</td></tr><tr><td>37 &amp;deg;C incubator for bacteria</td><td></td><td></td><td>For bacterial transformations and initial overnight growth of large cultures of Rosetta2 &lt;em&gt;E. coli&lt;/em&gt; transformed with TriEx4-DHX36</td></tr><tr><td>37 &amp;deg;C/14&amp;nbsp;&amp;deg;C shaking incubator for bacteria</td><td></td><td></td><td>For growth and protein induction of large cultures of Rosetta2 &lt;em&gt;E. coli&lt;/em&gt; 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&amp;nbsp;</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&amp;nbsp;</td></tr></tbody></table>

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

고차 핵산 구조는 모두 DNA와 RNA의 구아닌이 풍부한 지역에서 형성 할 수있는 G-quadruplexes (G4를, G4 구조)라는 높은 열적으로 안정하다. 이 인간 게놈에&gt; 375,000 추정 G4 형성 서열이 있고, 그들은 프로모터 영역, 번역되지 않은 지역 (UTRs)에 충실하고, 텔로미어 반복 내에서. 때문에 이러한 구조는 복제 및 전사와 같은 세포 과정에 영향을 미칠 가능성이있는 경우에, 셀들을 관리하는 효소를 진화했다. 하나의 효소는 생화학 실험실 및 나가미네 등에 의해 공동으로 특징이었다 G4 Resolvase 1 (G4R1)입니다. 매우 긴밀하게 (낮은 오후 범위에서 K d 개) 모두 G4-DNA와 G4-RNA에 결합하는 것으로. G4R1는 헬라 세포 용 해물에서 G4-해결 활동의 대부분의 원인과 이후 텔로미어 대사, 림프 개발, 유전자 전사, 조혈 및 면역 감시의 역할을 내포하고있다. EF 할 수있는 능력분히 표현하고 촉매 활성 G4R1는 G4의 구조와 G4-해결 효소의 운동의 상호 작용에 더 통찰력을 얻기에 관심이 실험실을 위해 중요하다 정화. 여기, 우리는 재조합 G4R1 (rG4R1)의 정제에 대한 자세한 방법을 설명합니다. 설명 된 절차는 C- 말단 히스티딘 표지 된 효소의 전통적인 선호도 기반 정제 바인딩 및 ATP- 고도로 활성 효소를 정제 G4-DNA를 긴장을 rG4R1 능력의 이용 인간 코돈 최적화 된 세균의 발현 포함 종속 용출 단계. 프로토콜은 또한 rG4R1의 효소 활성 G4-DNA를 푸는 정제 효소의 능력을 조사하여 측정하는 품질 관리 단계를 포함한다. 방법도 정제 rG4R1의 정량화를 허용 것이 기재되어있다. 이 프로토콜의 대체 적응이 논의된다.

이 프로토콜 (그림 1)는 일상적으로 거의 순수, 촉매 활성 rG4R1을 산출한다. rG4R1 0.013 μL 범위 (도 위에서 설명한 G4 활성 분석으로 분석으로서, - TAMRA 표지 tetramolecular G4-DNA 0.2 pmol의 50 %가 0.2 이내에 단량체로 변환되는 효소 활성의 척도로서, 이는 전형적으로 관찰 2). 정제 rG4R1의 쿠마 염색은 G4-DNA 구슬을 결합 할 수 및 ATP 의존에서 용출 능력을 유지하고 그 rG4R1...

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 Resolvase1는 G4 결합 단백질에 대한 소감보고 된 친화 G-quadruplex (G4) 구조에 결합 HeLa 세포에서 G4-DNA 풀기 활동의 대부분을 나타냅니다. 우리는 특히 촉매 활성 재조합 G4R1를 정화 친 화성 및 G4-Resolvase1의 ATP 의존 풀기 활동을 마구를 채우는 새로운 프로토콜을 설명합니다.

효소 활성 G4 Resolvase1의 정화를위한 G-quadruplex DNA 화성 접근

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 ...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

9.5K 조회수.  Wake Forest School of Medicine.  G4 Resolvase1는 G4 결합 단백질에 대한 소감보고 된 친화 G-quadruplex (G4) 구조에 결합 HeLa 세포에서 G4-DNA 풀기 활동의 대부분을 나타냅니다. 우리는 특히 촉매 활성 재조합 G4R1를 정화 친 화성 및 G4-Resolvase1의 ATP 의존 풀기 활동을 마구를 채우는 새로운 프로토콜을 설명합니다. 

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

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.

에서 외부 막 소포 내에서 감독 단백질 포장<em&gt; 대장균</em&gt; : 디자인, 생산 및 정제

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

연구

생화학

Tools to Study the Role of Architectural Protein HMGB1 in the Processing of Helix Distorting, Site-specific DNA Interstrand Crosslinks

High mobility group box 1 (HMGB1) protein is a non-histone architectural protein that is involved in regulating many important functions in the genome, such as transcription, DNA replication, and DNA repair. HMGB1 binds to structurally distorted DNA with higher affinity than to canonical B-DNA. For example, we found that HMGB1 binds to DNA interstrand crosslinks (ICLs), which covalently link the two strands of the DNA, cause distortion of the helix, and if left unrepaired can cause cell death. Due to their cytotoxic potential, several ICL-inducing agents are currently used as chemotherapeutic agents in the clinic. While ICL-forming agents show preferences for certain base sequences (e.g., 5&#39;-TA-3&#39; is the preferred crosslinking site for psoralen), they largely induce DNA damage in an indiscriminate fashion. However, by covalently coupling the ICL-inducing agent to a triplex-forming oligonucleotide (TFO), which binds to DNA in a sequence-specific manner, targeted DNA damage can be achieved. Here, we use a TFO covalently conjugated on the 5&#39; end to a 4&#39;-hydroxymethyl-4,5&#39;,8-trimethylpsoralen (HMT) psoralen to generate a site-specific ICL on a mutation-reporter plasmid to use as a tool to study the architectural modification, processing, and repair of complex DNA lesions by HMGB1 in human cells. We describe experimental techniques to prepare TFO-directed ICLs on reporter plasmids, and to interrogate the association of HMGB1 with the TFO-directed ICLs in a cellular context using chromatin immunoprecipitation assays. In addition, we describe DNA supercoiling assays to assess specific architectural modification of the damaged DNA by measuring the amount of superhelical turns introduced on the psoralen-crosslinked plasmid by HMGB1. These techniques can be used to study the roles of other proteins involved in the processing and repair of TFO-directed ICLs or other targeted DNA damage in any cell line of interest.

Targeted DNA damage can be achieved by tethering a DNA damaging agent to a triplex-forming oligonucleotide (TFO). Using modified TFOs, DNA damage-specific protein association, and DNA topology modification can be studied in human cells by the utilization of modified chromatin immunoprecipitation assays and DNA supercoiling assays described herein.

도구 나선 왜곡, 사이트 별 DNA Interstrand 가교의 처리에 건축 단백질 HMGB1의 역할을 연구하는

High mobility group box 1 (HMGB1) protein is a non-histone architectural protein that is involved in regulating many important functions in the genome, ...

유전학

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 replisome is one of the simplest replication systems known, composed of only four proteins, which is an attractive feature for biochemical experiments. Special emphasis is placed on the synthesis of ribonucleotide primers by the T7 primase-helicase, which are used by DNA polymerase to initiate DNA synthesis. Because the mechanisms of DNA replication are conserved across evolution, these protocols should be applicable, or useful as a conceptual springboard, to investigators using other model systems. The protocols described here are highly sensitive and an experienced investigator can perform these experiments and obtain data for analysis in about a day. The only specialized piece of equipment required is a rapid-quench flow instrument, but this piece of equipment is relatively common and available from various commercial sources. The major drawbacks of these assays, however, include the use of radioactivity and the relative low throughput.

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

We describe sensitive, gel-based discontinuous assays to examine the kinetics of lagging-strand initiation using the replication proteins of bacteriophage T7.

보온재 가닥 DNA 합성의 속도론<em&gt; 체외</em&gt;는 박테리오파지 T7 복제 단백질에 의해

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 ...

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA processing, and telomere elongation. During these processes, various proteins bind and resolve G4 structures to perform their function. As the function of G4 often depends on the stability of its folded structure, it is important to investigate how G4 binding proteins regulate the stability of G4. This work presents a method to manipulate single G4 molecules using magnetic tweezers, which enables studies of the regulation of G4 binding proteins on a single G4 molecule in real time. In general, this method is suitable for a wide scope of applications in studies for proteins/ligands interactions and regulations on various DNA or RNA secondary structures.

A single-molecule magnetic tweezers platform to manipulate G-quadruplexes is reported, which allows for the study of G4 stability and regulation by various proteins.

마그네틱 핀셋으로 G-quadruplexes의 단일 분자 조작

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA ...

Chemical Mapping을 사용하여 DNA 템플릿에서 G-Quadruplexes 식별

In Vitro Chemical Mapping of G-Quadruplex DNA Structures by Bis-3-Chloropiperidines

G-quadruplexes (G4s) are biologically relevant, non-canonical DNA structures that play an important role in gene expression and diseases, representing significant therapeutic targets. Accessible methods are required for the in vitro characterization of DNA within potential G-quadruplex-forming sequences (PQSs). B-CePs are a class of alkylating agents that have proven to be useful chemical probes for investigation of the higher-order structure of nucleic acids. This paper describes a new chemical mapping assay exploiting the specific reactivity of B-CePs with the N7 of guanines, followed by direct strand cleavage at the alkylated Gs. 
Namely, to distinguish G4 folds from unfolded DNA forms, we use B-CeP 1 to probe the thrombin-binding aptamer (TBA), a 15-mer DNA able to assume the G4 arrangement. Reaction of B-CeP-responding guanines with B-CeP 1 yields products that can be resolved by high-resolution polyacrylamide gel electrophoresis (PAGE) at a single-nucleotide level by locating individual alkylation adducts and DNA strand cleavage at the alkylated guanines. Mapping using B-CePs is a simple and powerful tool for the in vitro characterization of G-quadruplex-forming DNA sequences, enabling the precise location of guanines involved in the formation of G-tetrads.

저자 스포트라이트: Bis-3-Chloropiperidine 기반 화학 매핑에 의한 DNA G-Quadruplex 특성화 

Bis-3-chloropiperidines (B-CePs) are useful chemical probes to identify and characterize G-quadruplex structures in DNA templates in vitro. This protocol details the procedure to perform probing reactions with B-CePs and to resolve reaction products by high-resolution polyacrylamide gel electrophoresis.

In Vitro (시험관) Bis-3-Chloropiperidines에 의한 G-Quadruplex DNA 구조의 화학적 매핑

G-quadruplexes (G4s) are biologically relevant, non-canonical DNA structures that play an important role in gene expression and diseases, representing ...

<meta charset="utf8"></head><body>His-tagged FEN1 단백질의 효율적인 정제를 위한 FPLC 최적화

Affinity Purification of a 6X-His-Tagged Protein using a Fast Protein Liquid Chromatography System

Functional characterization of proteins requires them to be expressed and purified in substantial amounts with high purity to perform biochemical assays. The Fast Protein Liquid Chromatography (FPLC) system allows high-resolution separation of complex protein mixtures. By adjusting various parameters in FPLC, such as selecting the appropriate purification matrix, regulating the protein sample's temperature, and managing the sample's flow rate onto the matrix and the elution rate, it is possible to ensure the protein's stability and functionality. In this protocol, we will demonstrate the versatility of the FPLC system to purify 6X-His-tagged flap endonuclease 1 (FEN1) protein, produced in bacterial cultures. To improve protein purification efficiency, we will focus on multiple considerations, including proper column packing and preparation, sample injection using a sample loop, flow rate of sample application to the column, and sample elution parameters. Finally, the chromatogram will be analyzed to identify fractions containing high yields of protein and considerations for proper recombinant protein long-term storage. Optimizing protein purification methods is crucial for improving the precision and reliability of protein analysis.

<meta charset="utf8"></head><body>저자 스포트라이트: Optimizing Affinity Chromatography for His-Tagged FEN1 Protein

This article provides a procedure for the affinity purification of a human recombinant protein, flap endonuclease 1 (FEN1), which has been labeled with a 6X-histidine tag. The protocol involves the utilization of two distinct immobilized metal ion columns for the purification of the tagged protein.

Fast Protein 액체 크로마토그래피 시스템을 사용한 6X-His-Tagged 단백질의 친화성 정제 

Functional characterization of proteins requires them to be expressed and purified in substantial amounts with high purity to perform biochemical assays. ...

효소 활성 G4 Resolvase1의 정화를위한 G-quadruplex DNA 화성 접근

요약

더 많은 비디오 탐색

이 비디오의 챕터

에서 외부 막 소포 내에서 감독 단백질 포장

도구 나선 왜곡, 사이트 별 DNA Interstrand 가교의 처리에 건축 단백질 HMGB1의 역할을 연구하는

보온재 가닥 DNA 합성의 속도론

마그네틱 핀셋으로 G-quadruplexes의 단일 분자 조작

In Vitro (시험관) Bis-3-Chloropiperidines에 의한 G-Quadruplex DNA 구조의 화학적 매핑

In Vitro (시험관) Bis-3-Chloropiperidines에 의한 G-Quadruplex DNA 구조의 화학적 매핑

In Vitro (시험관) Bis-3-Chloropiperidines에 의한 G-Quadruplex DNA 구조의 화학적 매핑

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Fast Protein 액체 크로마토그래피 시스템을 사용한 6X-His-Tagged 단백질의 친화성 정제

Fast Protein 액체 크로마토그래피 시스템을 사용한 6X-His-Tagged 단백질의 친화성 정제