Name: Overexpressie en zuivering van een toxische nuclease van Escherichia coli (Video) | JoVE Journal
Uploaded: 2025-08-29T14:00:00.000+00:00
Duration: 8 min 13 s
Description: Leer de overexpressie en zuivering van toxische nucleases zoals Nsp15 uit Escherichia coli met affiniteits- en grootte-exclusiechromatografie. Deze methode biedt een betrouwbaar kader voor biochemisch onderzoek naar virale eiwitten.

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</ol>

<table><tbody><tr><td>0.22 &micro;m PES, Sterilizing, Low Protein Binding, Filter System, 1 L</td><td>Corning</td><td>431098</td><td></td></tr><tr><td>AEBSF serine protease inhibitor</td><td>Goldbio</td><td>30827-99-7</td><td></td></tr><tr><td>Agar, bacteriological</td><td>Amresco</td><td>J637</td><td></td></tr><tr><td>&Auml;KTA go purification system</td><td>Cytiva</td><td>29383015</td><td></td></tr><tr><td>Amicon Ultra 15 mL, 30 kDa MWCO concentrator</td><td>Millipore</td><td>UFC903024</td><td></td></tr><tr><td>Ampicillin sodium</td><td>Goldbio</td><td>A-301-25</td><td></td></tr><tr><td>Baffled shake flasks</td><td>Nalgene&nbsp;</td><td>F0527</td><td></td></tr><tr><td>Benchtop centrifuge</td><td>Eppendorf&nbsp;</td><td>5920R</td><td></td></tr><tr><td>BioPhotometer plus</td><td>Eppendorf</td><td>E-BP6132</td><td></td></tr><tr><td>Bovine thrombin</td><td>BioPharm</td><td>91-030</td><td></td></tr><tr><td>Bradford dye reagent</td><td>Boston BioProducts</td><td>BPA-200</td><td></td></tr><tr><td>Bromophenol blue</td><td>Sigma-Aldrich</td><td>114931</td><td></td></tr><tr><td>Calcium Chloride</td><td>Sigma-Aldrich</td><td>C4901</td><td></td></tr><tr><td>Digital dry bath</td><td>Bio Rad</td><td>1660562</td><td></td></tr><tr><td>Disposable spreaders, L-shaped</td><td>MedSupply Partners</td><td>62-1014-1</td><td></td></tr><tr><td>Dithiothreitol</td><td>Goldbio</td><td>27565-41-9</td><td></td></tr><tr><td>DNaseI from bovine pancrease</td><td>Sigma-Aldrich</td><td>D4527</td><td></td></tr><tr><td>Douce tissue grinder, 40 mL</td><td>Wheaton</td><td>357546</td><td></td></tr><tr><td>Econo-column chromagtography columns</td><td>Bio-Rad</td><td>737-1532</td><td></td></tr><tr><td>Enduro minimix nutating mixer</td><td>Labnet</td><td>S0600</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>459844</td><td></td></tr><tr><td>Excella E25R Inc/Ref Shaker</td><td>Eppendorf</td><td>NB-E25R</td><td></td></tr><tr><td>FastPette V2</td><td>Labnet</td><td>P2000</td><td></td></tr><tr><td>Fiberlite F10-4x1000 LEX fixed angle rotor</td><td>Thermo Fisher Scientific</td><td>096-041075</td><td></td></tr><tr><td>Fiberlite F21-8x50y fixed angle rotor</td><td>Thermo Fisher Scientific</td><td>096-084275</td><td></td></tr><tr><td>Gel Doc EZ Imager</td><td>Bio-Rad</td><td>1708270</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Alrich</td><td>G5516</td><td></td></tr><tr><td>Glycine</td><td>Bio-Rad</td><td>161-0718</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Hot plate stirrer</td><td>VWR</td><td>12365-346</td><td></td></tr><tr><td>Hydrochloric acid</td><td>VWR</td><td>BDH3028</td><td></td></tr><tr><td>Imidazole</td><td>Goldbio</td><td>I-902-25</td><td></td></tr><tr><td>Isopropyl-&szlig;-D-thiogalactioside (IPTG)</td><td>Goldbio</td><td>12481C25</td><td></td></tr><tr><td>Lemo21(DE3) competent E. coli cells</td><td>New England Biolabs</td><td>C2528J</td><td></td></tr><tr><td>Manganese(II) chloride tetrahydrate</td><td>Sigma-Aldrich</td><td>M3634</td><td></td></tr><tr><td>Microcentrifuge tubes, 0.5 mL</td><td>VWR</td><td>87003-290</td><td></td></tr><tr><td>Microcentrifuge tubes, 1.7 mL</td><td>MedSupply Partners</td><td>15-1151</td><td></td></tr><tr><td>Microcentrifuge tubes, 2 mL</td><td>Axygen</td><td>MCT-200-C</td><td></td></tr><tr><td>Mini-PROTEAN tetra vertical electrophoresis cell</td><td>Bio-Rad</td><td>10025025</td><td></td></tr><tr><td>Mini-Protean Tris-glycine gels, 4-20%</td><td>Bio-Rad</td><td>4568094</td><td></td></tr><tr><td>MyFuge mini centrfuge</td><td>MedSupply Partners</td><td>C1008-B</td><td></td></tr><tr><td>Nalgene 1 L super-speed centrifuge bottle</td><td>Thermo Fisher Scientific</td><td>3141-1006</td><td></td></tr><tr><td>Nalgene oak ridge centrifuge tube</td><td>Thermo Fisher Scientific</td><td>31139-0050</td><td></td></tr><tr><td>Nanophotometer</td><td>Implen</td><td>3769</td><td></td></tr><tr><td>Non-shaking incubator</td><td>Labnet</td><td>I 5110&nbsp;</td><td></td></tr><tr><td>Overexpress C41(DE3) chemically competent cells</td><td>Biosearch Technologies</td><td>60442-1</td><td></td></tr><tr><td>Overexpress C41(DE3) chemically competent cells</td><td>Sigma-Aldrich</td><td>CMC0017</td><td>Discontinued; Biosearch C41(DE3) cells or NEB Lemo21 cells are an alternative</td></tr><tr><td>Petri dishes</td><td>MedSupply Partners</td><td>62-1077-3</td><td></td></tr><tr><td>Plasmids (pET-14b backbone)</td><td>Genscript</td><td>Custom order</td><td></td></tr><tr><td>Plastic micro-cuvettes</td><td>Fisher Scientific</td><td>14955127</td><td></td></tr><tr><td>PowerPacuniversal power supply</td><td>Bio-Rad</td><td>1645070</td><td></td></tr><tr><td>Precision glide needle</td><td>Becton Dixon</td><td>305198</td><td></td></tr><tr><td>Precision plus protein unstained standards</td><td>Bio-Rad</td><td>1610363</td><td></td></tr><tr><td>Recovery medium for expression&nbsp;</td><td>Sigma-Aldrich</td><td>CMR0001K</td><td></td></tr><tr><td>S-4x1000 swinging bucket rotor</td><td>Eppendorf</td><td>EP-S4X1</td><td></td></tr><tr><td>Serological pipette, 10 mL</td><td>Faclon</td><td>357551</td><td></td></tr><tr><td>Serological pipette, 25 mL</td><td>Faclon</td><td>35725</td><td></td></tr><tr><td>Serological pipette, 5 mL</td><td>Faclon</td><td>35743</td><td></td></tr><tr><td>Serological pipette, 50 mL</td><td>Pr1ma</td><td>PR-Sero-50</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S9888</td><td></td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sigma-Aldrich</td><td>L3771</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>567530</td><td></td></tr><tr><td>Sonic dismembrator</td><td>Fisher Scientific</td><td>FB505</td><td></td></tr><tr><td>Sorvall Lynx 4000 Centrifuge</td><td>Thermo Fisher Scientific</td><td>75006580</td><td></td></tr><tr><td>Spephadex G-25 M PD-10 column</td><td>Cytiva</td><td>17085101</td><td></td></tr><tr><td>&szlig;-Mercaptoethanol</td><td>Millipore</td><td>444203</td><td></td></tr><tr><td>Stainless steele Griffin beaker</td><td>Polar Ware</td><td>13977-010-EA</td><td></td></tr><tr><td>Sterile Falcon tubes, 15 mL</td><td>Falcon</td><td>352097</td><td></td></tr><tr><td>Sterile Falcon tubes, 50 mL</td><td>Falcon</td><td>352098</td><td></td></tr><tr><td>Superdex 200 10/300 GL column</td><td>Cytiva</td><td>28990944</td><td></td></tr><tr><td>Syringes, 1 mL</td><td>Air-Tite&nbsp;</td><td>A1</td><td></td></tr><tr><td>Syringes, 3 mL</td><td>Becton Dixon</td><td>309657</td><td></td></tr><tr><td>TAL

overexpressie en zuivering van een toxische nuclease van escherichia coli

Overexpressing and Purifying a Toxic Nuclease from Escherichia coli

Overexpressie en zuivering van een toxische nuclease van Escherichia coli

Escherichia coli has been widely employed as an expression system to produce recombinant proteins. Yet some types of recombinant proteins, such as ...

overexpressing-and-purifying-a-toxic-nuclease-from-escherichia-coli

Research

JoVE Journal

Biochemistry

Biochemie

Eiwit transductie maakt de directe levering van biologisch actieve eiwitten in cellen. In tegenstelling tot conventionele methoden, zoals DNA-transfectie of virale transductie deze niet-invasieve paradigma maakt zeer efficiënte cellulaire manipulatie in een titreerbare manier te omzeilen cellulaire toxiciteit en het risico van oncogene transformatie door permanente genetische modificatie.

Technische Cel-permeabel Protein

Systematische, grootschalige synthetische genetische (gen-gen of epistase) interactie schermen kunnen worden gebruikt om genetische redundantie en route overspraak verkennen. Hier hebben we een high-throughput kwantitatieve synthetische genetische scala screening technologie te beschrijven, genoemd eSGA door ons ontwikkelde voor het ophelderen van epistatisch relaties en het verkennen van genetische interactie netwerken...

Het in kaart brengen Bacteriële Functionele Netwerken en Wegen in<em&gt; Escherichia Coli</em&gt; Gebruik van synthetische Genetische Arrays

Exonucleasen spelen een cruciale rol bij het waarborgen van de stabiliteit van het genoom. Verlies van de WRN-exonucleasefunctie resulteert in voortijdige veroudering. Het bestuderen van substraten en andere vereisten van de nuclease in vitro kan helpen bij het verduidelijken van zijn rol in vivo. Hier demonstreren we een snelle en reproduceerbare fluorescentie-gebaseerde assay om zijn nuclease-activiteit te meten.

Een fluorescentie-gebaseerde Exonuclease Assay te karakteriseren DmWRNexo, ortholoog van Human Progeroid WRN Exonuclease, en de toepassing ervan op andere Nucleasen

Deze vijfdaagse protocol beschrijft alle stappen, apparatuur en aanvullende software voor het maken en uitvoeren van een efficiënte endogene Escherichia coli op basis TX-TL-cel-vrije expressie systeem vanaf nul. Met reagentia, het protocol duurt 8 uur of minder voor het inrichten van een reactie, verzamelen en verwerken van gegevens.

Protocollen voor implementatie van een Escherichia coli Op basis TX-TL Cell-Free Expression System for Synthetic Biology

Eiwit co-expressie is een krachtige alternatief voor de reconstitutie in vitro van eiwitcomplexen, en helpt bij het uitvoeren van biochemische en genetische tests in vivo. Hier rapporteren we over het gebruik van eiwit co-expressie in Escherichia coli om eiwitcomplexen te verkrijgen en om de mutatiefrequentie van cellen af te stemmen.

De veelzijdige voordelen van Protein Co-expressie in<em&gt; Escherichia coli</em

Een hoge opbrengst Werkwijze voor eenstaps negatieve zuivering van recombinant Helicobacter pylori-neutrofiel activerend eiwit (HP-NAP) tot overexpressie gebracht in Escherichia coli door diethylaminoethyl harsen batchmodus is beschreven. HP-NAP gezuiverd met deze methode is gunstig voor de ontwikkeling van vaccins, geneesmiddelen of diagnostica voor H. pylori-geassocieerde ziekten.

Een stap Negatieve Chromatografische zuivering van<em&gt; Helicobacter pylori</em&gt; Neutrofiel-activerend eiwit overexpressie in<em&gt; Escherichia coli</em&gt; In Batchmodus

Een eenvoudig protocol voor overexpressie en zuivering van codon-geoptimaliseerde, humane cis -prenyltransferase, onder niet-denaturerende omstandigheden, van Escherichia   Coli , wordt beschreven, samen met een enzymatische activiteitstest. Dit protocol kan worden genegaliseerd voor de productie van andere cis- prenyltransferase eiwitten in hoeveelheid en kwaliteit geschikt voor mechanistische studies.

Overexpressie en zuivering van het menselijk Cis -prenyltransferase in Escherichia coli

Hier presenteren we een protocol voor het gebruik van reeds bestaande antibiotica resistentie-cassette schrapping constructies als basis voor het maken van schrapping mutanten in andere stammen van E. coli . Dergelijke mutaties schrapping kunnen worden gemobiliseerd en ingevoegd in de corresponderende locus van een geadresseerde stam met behulp van P1 bacteriofaag transductie.

Productie van Gene verwijderingen in Escherichia coli door P1 transductie met accijnsplichtige antibioticaresistentie Cassettes

Protocatechuate 3, 4-dioxygenase (PCD) kan enzymatisch vrij diatomaire zuurstof verwijderen uit een watersysteem met behulp van de substraat protocatechuïnezuur zuur (PCA). Dit protocol beschrijft de expressie, zuivering, en activiteit analyse van deze enzym voor het opruimen van zuurstof.

Expressie en zuivering van Nuclease-vrije zuurstof Scavenger Protocatechuate 3, 4-dioxygenase

De endonucleases van de beperking met nieuwe opeenvolgingsspecificiteit kunnen van enzymen worden ontwikkeld die een gedeeltelijk gedegenereerde opeenvolging erkennen. Hier bieden we een gedetailleerd protocol dat we met succes gebruikt om de volgorde specificiteit van NlaIV enzym te veranderen. De belangrijkste ingrediënten van het protocol zijn de in vitro compartimentering van de transcriptie/vertaalreactie en selectie van varianten met nieuwe sequentiespecifiekekenmerken.

In Vitro Gerichte Evolutie van een beperking Endonuclease met strengere specificiteit

Bacteriën scheiden nanometergrote extracellulaire blaasjes (EV's) af die bioactieve biologische moleculen dragen. EV-onderzoek richt zich op het begrijpen van hun biogenese, rol in microbe-microbe en gastheer-microbe interacties en ziekte, evenals hun potentiële therapeutische toepassingen. Een workflow voor schaalbare isolatie van EV's van verschillende bacteriën wordt gepresenteerd om standaardisatie van EV-onderzoek te vergemakkelijken.

Schaalbare isolatie en zuivering van extracellulaire blaasjes van Escherichia coli en andere bacteriën

Leer de overexpressie en zuivering van toxische nucleases zoals Nsp15 uit Escherichia coli met affiniteits- en grootte-exclusiechromatografie. Deze methode biedt een betrouwbaar kader voor biochemisch onderzoek naar virale eiwitten.

Overexpressie en zuivering van een toxische nuclease van Escherichia coli

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