Name: detection and removal of nuclease contamination during purification of recombinant prototype foamy virus integrase
Uploaded: 2017-12-08T14:00:00
Description: Watch this Scientific Journal Video about Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase at JoVE.com

Dette arbejde blev støttet af NIH AI099854 og AI126742 til nøglen.

1. fremkalde PFV i udtryk i E. coli
<ol><li>
Tilføje 1 μL PFV IN udtryk plasmid (10 ng/μl) til 20 μL af E. coli BL21(DE3) pLysS (20 μL alikvot af kommercielt tilgængelige celler har > 2 x 106 CFU/μg plasmid) i 1,5 mL rør. Bland forsigtigt af finger trykke eller svippede røret. Ruger på is 5 min.
	<ol>
<li>Heat shock for præcis 30 s i en 42 ˚C vand bad og derefter straks tilbage til is i 2 min.</li>
		<li>Tilsættes 80 μl stuetemperatur super o...

<ol>
	<li>Lopez, M. A., Mackler, R. M., Yoder, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+nuclease+contamination+during+purification+of+recombinant+prototype+foamy+virus+integrase.">Removal of nuclease contamination during purification of recombinant prototype foamy virus integrase.</a> J Virol Methods. 235, 134-138 (2016).</li><li>Senavirathne, G., 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=Widespread+nuclease+contamination+in+commonly+used+oxygen-scavenging+systems.">Widespread nuclease contamination in commonly used oxygen-scavenging systems.</a> Nat Methods. 12 (10), 901-902 (2015).</li><li>Gunn, K. H., Marko, J. F., Mondragon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+orthogonal+single-molecule+experiment+reveals+multiple-attempt+dynamics+of+type+IA+topoisomerases.">An orthogonal single-molecule experiment reveals multiple-attempt dynamics of type IA topoisomerases.</a> Nat Struct Mol Biol. 24 (5), 484-490 (2017).</li><li>Valkov, E., 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=Functional+and+structural+characterization+of+the+integrase+from+the+prototype+foamy+virus.">Functional and structural characterization of the integrase from the prototype foamy virus.</a> Nucleic Acids Res. 37 (1), 243-255 (2009).</li><li>Coffin, J. M., Hughes, S. H., Varmus, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Retroviruses. , (1997).</li><li>Li, M., Craigie, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+of+viral+DNA+ends+channels+the+HIV-1+integration+reaction+to+concerted+integration.">Processing of viral DNA ends channels the HIV-1 integration reaction to concerted integration.</a> J Biol Chem. 280 (32), 29334-29339 (2005).</li><li>Li, M., Mizuuchi, M., Burke, T. R., Craigie, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+DNA+integration:+reaction+pathway+and+critical+intermediates.">Retroviral DNA integration: reaction pathway and critical intermediates.</a> EMBO J. 25 (6), 1295-1304 (2006).</li><li>Sinha, S., Grandgenett, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+human+immunodeficiency+virus+type+1+integrase+exhibits+a+capacity+for+full-site+integration+in+vitro+that+is+comparable+to+that+of+purified+preintegration+complexes+from+virus-infected+cells.">Recombinant human immunodeficiency virus type 1 integrase exhibits a capacity for full-site integration in vitro that is comparable to that of purified preintegration complexes from virus-infected cells.</a> J Virol. 79 (13), 8208-8216 (2005).</li><li>Goodarzi, G., Im, G. J., Brackmann, K., Grandgenett, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concerted+integration+of+retrovirus-like+DNA+by+human+immunodeficiency+virus+type+1+integrase.">Concerted integration of retrovirus-like DNA by human immunodeficiency virus type 1 integrase.</a> J Virol. 69 (10), 6090-6097 (1995).</li><li>Vora, A. C., Grandgenett, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+catalytic+properties+of+retrovirus+integrase-DNA+complexes+capable+of+efficiently+performing+concerted+integration.">Assembly and catalytic properties of retrovirus integrase-DNA complexes capable of efficiently performing concerted integration.</a> J Virol. 69 (12), 7483-7488 (1995).</li><li>Jones, N. 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=Retroviral+intasomes+search+for+a+target+DNA+by+1D+diffusion+which+rarely+results+in+integration.">Retroviral intasomes search for a target DNA by 1D diffusion which rarely results in integration.</a> Nat Commun. 7, 11409 (2016).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+gel+electrophoresis+for+the+separation+of+DNA+fragments.">Agarose gel electrophoresis for the separation of DNA fragments.</a> J Vis Exp. (62), (2012).</li></ol>

Rekombinante proteiner, der interagerer med DNA, såsom DNA reparere proteiner, ilt skraldemanden for enkelt molekyle mikroskopi applikationer eller retroviral integrases, bør være fri for kontaminerende bakteriel nukleaser2,3. Disse forurenende stoffer kan forvirre fortolkning af resultater under bulk biokemiske eller enkelt molekyle assays.
Vi har fundet, at en bakteriel nukleasen ofte Co renser med PFV IN. Imidlertid viser PFV i e...

Biokemiske og enkelt molekyle studier af protein interaktion med DNA kræver usædvanlig ren rekombinante proteiner. Forurener nukleaser fra bakterier kan sløre resultaterne af disse analyser. En kontaminerende nukleasen er blevet fundet i forberedelserne af rekombinante proteiner ilt scavenger protocatechuate-3,4-dioxygenase (PCD) og prototype skummende virus (PFV) integrase (IN) isoleret fra Escherichia coli (E. coli)1 , 2 , 3.
Retroviral integration assays stole på konvertering af supercoiled DNA til hakker eller lineær produkter som en foranstaltning af aktivitet4. Under cellulære infektion tiltræder IN de to ender af en viral cDNA-vært kromatin5. Hver ende tiltræder reaktion kaldes strand overførsel. Assays af rekombinant IN aktivitet kan deltage to DNA oligomerer efterligne den viral cDNA slutter til en target-DNA i en samordnet integration reaktion4,5,6,7,8. Alternativt optages rekombinant IN kun en DNA ende i en ikke-fysiologisk relevante halv site integration reaktion9,10. Når supercoiled plasmid DNA er målet for integration, samordnet integration produkter er lineariseret DNA og halv site integration produkter er afslappet cirkler. Disse reaktionsprodukter identificeres af deres relative mobilitet under Agarosen gel elektroforese1. Hvis den rekombinante i har en kontaminerende nukleasen, vil der være spurious afslappet cirkler eller et eventuelt lineariseret plasmid forvirrende de eksperimentelle resultater. Viral DNA oligomerer kan mærkes fluorescently endegyldigt identificerer integration produkter, i modsætning til nukleasen produkter. Dog favoriserer i høj grad supercoiled DNA mål; tab af supercoiled plasmid til afslappet cirkler eller lineære DNA af kontaminerende nukleasen kunne skew resultaterne og fortolkningen af data11. Derfor er det bydende nødvendigt at fjerne bakteriel nukleaser fra retroviral i forberedelserne.
PFV i har en forskellig affinitet for heparin sepharose sammenlignet med bakteriel nukleasen1. PFV i og nukleasen kan adskilles ved en lineær gradient eluering fra heparin sepharose. Nukleasen registreres ikke let ved en ultraviolet (UV) absorbans ved 280 nm peak eller analytiske sodium dodecyl sulfat polyacrylamid gelelektroforese (SDS-PAGE). I stedet, nukleasen er opdaget af en nukleasen aktivitet assay beskæftiger konvertering af en supercoiled plasmid til afslappet cirkler eller lineær produkter. Hver fraktion efter heparin sepharose kromatografi er testet for nukleasen aktivitet. PFV i og nukleasen forurenende har ingen forskel i affinitet for Mono-Q anion harpiks. Der er en lille forskel i affinitet for Mono-S kation harpiks. Mono-S opløsning af bakteriel nukleasen og PFV IN giver imidlertid ikke effektiv adskillelse af proteiner. I sidste ende, heparin sepharose affinitet rensning tilbyder den bedste adskillelse af bakteriel nukleasen fra PFV i og har fordelen af ubegrænset belastning volumen.
Testning for kontaminerende nukleasen aktivitet kan tilpasses andre proteiner. Protein af interesse vil sandsynligvis have alternative affinitet egenskaber end PFV i; forskellen i bindende Karakteristik af protein af interesse og nukleasen forurenende stoffer skal bestemmes empirisk. Denne metode til at identificere nukleasen kontaminering kan tilpasses andre harpikser, herunder Mono-S kation eller Mono-Q anion exchange harpiks. Affinitet og ionbyttende harpikser kan tilbyde en pålidelig metode til at isolere en rekombinant protein af interesse fra forurener nukleaser med ingen begrænsninger for mængden af protein i kromatografi.

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			<td height="21" style="height:21px;width:216px;">BL21/DE3 Rosetta E. coli</td>
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			<td height="21" style="height:21px;width:216px;">LB broth</td>
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			<td height="21" style="height:21px;">IPTG&#160;</td>
			<td style="width:180px;">Denville Scientific</td>
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			<td height="21" style="height:21px;">ZnCl2</td>
			<td style="width:180px;">Sigma-Aldrich</td>
			<td style="width:119px;">208086</td>
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			<td height="21" style="height:21px;">Tris Ultra Pure</td>
			<td style="width:180px;">Gojira Fine Chemicals</td>
			<td style="width:119px;">UTS1003</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">NaCl</td>
			<td style="width:180px;">P212121</td>
			<td style="width:119px;">RP-S23020</td>
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			<td height="21" style="height:21px;">PMSF&#160;</td>
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			<td style="width:119px;">0754</td>
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			<td height="21" style="height:21px;">Imidazole&#160;</td>
			<td style="width:180px;">Sigma-Aldrich</td>
			<td style="width:119px;">I0250</td>
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			<td height="21" style="height:21px;">DTT&#160;</td>
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			<td height="21" style="height:21px;">UltraPure EDTA</td>
			<td style="width:180px;">Invitrogen/Gibco</td>
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			<td height="25" style="height:25px;">MgSO4</td>
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			<td style="width:119px;">0662</td>
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			<td height="21" style="height:21px;">Agarose&#160;</td>
			<td style="width:180px;">Denville Scientific</td>
			<td style="width:119px;">CA3510</td>
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			<td height="21" style="height:21px;">Ethidium bromide</td>
			<td style="width:180px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">BP1302</td>
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			<td height="21" style="height:21px;">Glycerol</td>
			<td style="width:180px;">Fisher Scientific</td>
			<td style="width:119px;">G37-20</td>
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			<td height="21" style="height:21px;">Ni-NTA Superflow</td>
			<td style="width:180px;">Qiagen</td>
			<td style="width:119px;">30430</td>
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			<td height="42" style="height:42px;">Heparin Sepharose 6 Fast Flow</td>
			<td style="width:180px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">17-0998-01</td>
			<td></td>
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			<td height="21" style="height:21px;">HRV14 3C protease&#160;</td>
			<td style="width:180px;">EMD Chemicals</td>
			<td style="width:119px;">71493-3</td>
			<td></td>
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detection and removal of nuclease contamination during purification of recombinant prototype foamy virus integrase

Integrase (IN) protein af retrovirus prototype skummende virus (PFV) er en model enzym til at studere mekanismen af retroviral integration. I forhold til IN fra andre retrovira, er PFV IN mere opløselige og mere medgørlige til eksperimentelle manipulation. Desuden, er det følsomme over for klinisk relevante human immundefekt virus (HIV-1) IN hæmmere, tyder på, at den katalytiske mekanisme for PFV i er svarer til at af HIV-1 i. IN katalyserer den kovalente sammenføjning af viral supplerende DNA (cDNA) til målet DNA i en proces kaldet strand overførsel. Denne strand overførsel reaktion introducerer rifter til target-DNA. Analyse af integration reaktionsprodukter kan blive forvirret af tilstedeværelsen af nukleaser, der ligeledes nick DNA. En bakteriel nukleasen har vist sig at co rense med rekombinante PFV IN udtrykt i Escherichia coli (E. coli). Her beskriver vi en metode til at isolere PFV i fra de kontaminerende nukleasen af heparin affinitet kromatografi. Fraktioner er let screenet for nukleasen kontaminering med supercoiled plasmid og Agarosen gelelektroforese. PFV i og de kontaminerende nukleasen vise alternative tilhørsforhold til heparin sepharose tillader en nukleasen-gratis forberedelse af rekombinante PFV i egnet til bulk biokemiske eller enkelt molekyle analyse af integration.

Rekombinante PFV IN er ofte forurenet med en bakteriel nukleasen1. Biokemiske integration assays afhænger kvantitering af konvertering af supercoiled plasmid DNA til afslappet cirkler og lineær produkter. Tilstedeværelsen af en forurenende nukleasen kunne føre til falske kvantitering af disse assays. Udtryk for PFV i med en hexahistidine tag er induceret i E. coli (figur 1) og først renset af nikkel affinitet kromatografi (figur 2</s...

Watch this Scientific Journal Video about Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase at JoVE.com

Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase

Rekombinant prototype skummende virus integrase protein er ofte forurenet med en bakteriel nukleasen under rensningen. Denne metode identificerer nukleasen forurening og fjerner det fra den endelige udarbejdelse af enzymet.

Registrering og fjernelse af nukleasen forurening under oprensning af rekombinante Prototype skummende Virus Integrase

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared ...

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