Name: detection of the ph-dependent activity of escherichia coli chaperone hdeb in vitro and in vivo
Uploaded: 2016-10-23T13:00:00
Description: Watch this Scientific Journal Video about Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo at JoVE.com

Vi takker Dr. Claudia Cremers for hendes nyttige råd om chaperone analyser. Ken Wan er anerkendt for sin tekniske bistand i HdeB rensning. Dette arbejde blev støttet af Howard Hughes Medical Institute (til JCAB) og National Institutes of Health tilskud RO1 GM102829 til JCAB og UJJ-UD understøttes af en postdoc stipendium fra den tyske Research Foundation (DFG).

1. Ekspression og oprensning af Periplasmisk HdeB BEMÆRK: HdeB blev udtrykt i E. coli-celler, der huser plasmidet pTrc- hdeB 10, og oprenset fra periplasmaet ved polymyxin lysis. <ol><li> Forbered en overnatning kultur af E. coli-celler, der huser plasmidet pTrc- hdeB 10 i 30 ml LB indeholdende 200 pg / ml ampicillin (LB Amp). Inokulere fire 1 L kulturer af LB Amp og dyrke dem ved 37 ° C og 200 rpm, indtil OD 600 nm p?...

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<html> For at undersøge mekanismen for aktivering og chaperone funktion af HdeB, store mængder HdeB skal udtrykkes og oprenses. Et antal ekspressionsvektorsystemer er tilgængelige til produktion af høje niveauer af et målprotein, herunder pTrc eller pBAD-vektorer, som begge blev anvendt i denne undersøgelse. Initiativtagerne er let tilgængelige for E. coli-RNA-polymerase og dermed give kraftigt opreguleret ekspression af HdeB i enhver E. coli-stamme. Dette aspekt er særligt relevant for in v...

En fælles naturlige miljø, hvor mikrobielle patogener oplever syre-induceret proteinudfoldning betingelser er den mammale maven (pH område 1-4), hvis sur pH tjener som en effektiv barriere mod fødevarebårne patogener 1. Proteinudfoldning og aggregering, som er forårsaget af aminosyresidekæde protonering, påvirker biologiske processer, skader cellulære strukturer og i sidste ende forårsager celledød 1,2. Da pH af den bakterielle periplasma ligevægt næsten øjeblikkeligt med den miljømæssige pH på grund af den frie diffusion af protoner gennem den porøse ydre membran, periplasmatiske og indre membranproteiner fra gramnegative bakterier er de mest sårbare cellulære komponenter under syre-stressbetingelser 3. For at beskytte deres periplasmatisk proteom mod hurtig syre-medieret skade, Gram-negative bakterier udnytte syre-aktiverede periplasmatiske chaperoner HdeA og HdeB. HdeA er en betinget uorganiseret chaperone <sup&gt; 4,5: ved neutral pH, HdeA er til stede som en foldet, chaperone-inaktiv dimer. Ved en pH-ændring under pH 3, er HdeA s chaperone-funktion hurtigt aktiveret 6,7. Aktivering af HdeA kræver dybtgående strukturelle ændringer, herunder dets dissociation i monomerer, og den delvise udfoldning af monomerer 6-8. Når den er aktiveret, HdeA binder til proteiner, der udfolder sig under sure betingelser. Det forhindrer effektivt deres aggregering både under inkubationen ved lav pH samt ved pH neutralisering. Ved tilbagevenden til pH 7,0, HdeA letter refoldning af sine klient proteiner i en ATP-uafhængig måde og konverterer tilbage til sin dimert chaperone-inaktiv form 9. Tilsvarende er den homologe chaperone HdeB også chaperone-inaktiv ved pH 7,0. I modsætning HdeA dog HdeB s chaperone aktivitet når sit tilsyneladende maksimum ved pH 4,0, under hvilke betingelser HdeB er stadig i vid udstrækning foldede og dimere 10. Desuden yderligere sænke pH causes inaktivering af HdeB. Disse resultater tyder på, at på trods af deres omfattende homologi, HdeA og HdeB forskellige i deres form for funktionel aktivering giver dem mulighed for at dække et bredt pH-område med deres beskyttende chaperone funktion. En anden chaperone, der er blevet impliceret i syreresistensen af E. coli er det cytoplasmatiske Hsp31, som synes at stabilisere ufoldede klient proteiner indtil neutrale betingelser er genoprettet. Den præcise tilstand af Hsp31 aktion, dog er forblevet gådefulde 12. I betragtning af, at andre tarmpatogene bakterier såsom salmonella mangler hdeAB operon, er det meget sandsynligt, at andre endnu uidentificerede periplasmatiske chaperoner kan findes, der er involveret i syre modstand af disse bakterier 11. Protokollerne præsenteres her tillader at overvåge pH-afhængig chaperonaktivitet af HdeB in vitro og in vivo 10 og kan anvendes til at undersøge andre chaperonersåsom Hsp31. Alternativt kan det komplekse net af transkriptionsfaktorer, der styrer ekspressionen af hdeAB potentielt undersøges af in vivo-stress assay. At karakterisere chaperone funktionen af proteiner in vivo, kan forskellige forsøgsopstillinger anvendes. En rute er at anvende protein udfoldning stresstilstande og fænotypisk karakterisering mutantstammer, der enten overudtrykker genet af interesse eller bærer en deletion af genet. Proteom undersøgelser kan udføres for at identificere, hvilke proteiner ikke længere aggregat under stressbetingelser når chaperonen er til stede, eller påvirkning af en chaperone på et specifikt enzym kan bestemmes under stressbetingelser anvendelse af enzymatiske assays 14-16. I denne undersøgelse valgte vi at overudtrykke HdeB i en rpoH deletion-stamme, som mangler varmechok sigma faktoren 32. RpoH styrer ekspressionen af alle større E. coli chaperones og sletning er kendt for at øge sensitivity for miljømæssige stress betingelser, der forårsager protein udfoldning 15. In vivo chaperonaktivitet af HdeB blev bestemt ved at overvåge dets evne til at undertrykke pH følsomheden af Δ rpoH-stamme. Alt i alt, de protokoller, der præsenteres her giver en hurtig og enkel fremgangsmåde til at karakterisere aktiviteten af et syreaktiveret chaperone in vitro såvel som in vivo sammenhæng.

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		<tr height="63">
			<td height="63" style="height:63px;width:216px;">NEB10-beta E. coli cells</td>
			<td style="width:64px;">New England Biolabs</td>
			<td style="width:119px;">C3019I</td>
			<td style="width:1791px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Ampicillin</td>
			<td style="width:64px;">Gold Biotechnology</td>
			<td style="width:119px;">A-301-3</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">LB Broth mix, Lennox</td>
			<td style="width:64px;">LAB Express</td>
			<td style="width:119px;">3003</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">IPTG</td>
			<td style="width:64px;">Gold Biotechnology</td>
			<td style="width:119px;">I2481C50</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Sodium chloride</td>
			<td style="width:64px;">Fisher Scientific</td>
			<td style="width:119px;">S271-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris</td>
			<td style="width:64px;">Amresco</td>
			<td style="width:119px;">0826-5kg</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">EDTA</td>
			<td style="width:64px;">Fisher Scientific</td>
			<td style="width:119px;">BP120-500</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Polymyxin B sulfate&#160;</td>
			<td style="width:64px;">ICN Biomedicals Inc.</td>
			<td style="width:119px;">100565</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">0.2 UM pore sterile Syringe Filter</td>
			<td style="width:64px;">Corning</td>
			<td style="width:119px;">431218</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">HiTrap Q HP (CV 5 ml)</td>
			<td style="width:64px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">17-1153-01</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Mini-Protean TGX, 15%</td>
			<td style="width:64px;">Bio-Rad</td>
			<td style="width:119px;">4561046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Malate dehydrogenase (MDH)</td>
			<td style="width:64px;">Roche</td>
			<td style="width:119px;">10127914001</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Potassium phosphate (Monobasic)</td>
			<td style="width:64px;">Fisher Scientific</td>
			<td style="width:119px;">BP362-500</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Potassium phosphate (Dibasic)</td>
			<td style="width:64px;">Fisher Scientific</td>
			<td style="width:119px;">BP363-1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">F-4500 fluorescence spectrophotometer</td>
			<td style="width:64px;">Hitachi</td>
			<td style="width:119px;">FL25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oxaloacetate</td>
			<td style="width:64px;">Sigma</td>
			<td style="width:119px;">O4126-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NADH</td>
			<td style="width:64px;">Sigma&#160;</td>
			<td style="width:119px;">N8129-100MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium phosphate monobasic</td>
			<td style="width:64px;">Sigma&#160;</td>
			<td style="width:119px;">S9390-2.5KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium phosphate dibasic</td>
			<td style="width:64px;">Sigma&#160;</td>
			<td style="width:119px;">S397-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lactate dehydrogenase (LDH)</td>
			<td style="width:64px;">Roche</td>
			<td style="width:119px;">10127230001</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Beckman Proteome Lab XL-I analytical Ultracentrifuge</td>
			<td style="width:64px;">Beckman Coulter</td>
			<td style="width:119px;">392764</td>
			<td>https://www.beckmancoulter.com/wsrportal/wsrportal.portal?_nfpb=true&#38;_windowLabel=UCM_RENDERER&#38;_urlType=render&#38;wlpUCM_RENDERER_path=%252Fwsr%252Fresearch-and-discovery%252Fproducts-and-services%252Fcentrifugation%252Fproteomelab-xl-a-xl-i%252Findex.htm#2/10//0/25/1/0/asc/2/392764///0/1//0/%2Fwsrportal%2Fwsr%2Fresearch-and-discovery%2Fproducts-and-services%2Fcentrifugation%2Fproteomelab-xl-a-xl-i%2Findex.htm/</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Centerpiece, 12 mm, Epon Charcoal-filled</td>
			<td style="width:64px;">Beckman Coulter</td>
			<td style="width:119px;">306493</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">AN-50 Ti Rotor, Analytical, 8-Place</td>
			<td style="width:64px;">Beckman Coulter</td>
			<td style="width:119px;">363782</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Wizard Plus Miniprep Kit</td>
			<td style="width:64px;">Promega</td>
			<td style="width:119px;">A1470</td>
			<td>used for plasmid purification (Protocol 5.1)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">L-arabinose</td>
			<td style="width:64px;">Gold Biotechnology</td>
			<td style="width:119px;">A-300-500</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Glycine</td>
			<td style="width:64px;">DOT Scientific Inc</td>
			<td style="width:119px;">DSG36050-1000</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Fluorescence Cell cuvette</td>
			<td style="width:64px;">Hellma Analytics</td>
			<td style="width:119px;">119004F-10-40</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Oligonucleotides</td>
			<td style="width:64px;">Invitrogen</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Phusion High-Fidelity DNA polymerase</td>
			<td style="width:64px;">New England Biolabs</td>
			<td style="width:119px;">M0530S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">dNTP set</td>
			<td style="width:64px;">Invitrogen</td>
			<td style="width:119px;">10297018</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Hydrochloric Acid</td>
			<td style="width:64px;">Fisher Scientific</td>
			<td style="width:119px;">A144-212</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Sodium Hydroxide</td>
			<td style="width:64px;">Fisher Scientific</td>
			<td style="width:119px;">BP359-500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Amicon Ultra 15 mL 3K NMWL</td>
			<td style="width:64px;">Millipore</td>
			<td style="width:119px;">UFC900324</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Centrifuge Avanti J-26XPI</td>
			<td style="width:64px;">Beckman Coulter</td>
			<td style="width:119px;">393127</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Varian Cary 50 spectrophotometer</td>
			<td style="width:64px;">Agilent Tech</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Spectra/Por 1 Dialysis Membrane MWCO: 6 kDa</td>
			<td style="width:64px;">Spectrum Laboratories</td>
			<td style="width:119px;">132650</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Amicon Ultra Centrifugal Filter Units 30K</td>
			<td style="width:64px;">Millipore</td>
			<td style="width:119px;">UFC803024</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">SDS</td>
			<td style="width:64px;">Fisher Scientific</td>
			<td style="width:119px;">bp166-500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Veriti 96-Well Thermal Cycler</td>
			<td style="width:64px;">Thermo Fisher</td>
			<td style="width:119px;">4375786</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of the ph-dependent activity of escherichia coli chaperone hdeb in vitro and in vivo

Bakterier ofte udsat for miljømæssige ændringer, såsom ændringer i pH, temperatur, redox status, lys eksponering eller mekanisk kraft. Mange af disse betingelser give protein udfolder sig i cellen og har skadelig indvirkning på overlevelsen af ​​organismen. En gruppe af ubeslægtede, stress-specifikke molekylære chaperoner er blevet vist at spille en væsentlig rolle i overlevelsen af ​​disse stressbetingelser. Mens fuldt foldet og chaperone-inaktiv, før stress, disse proteiner hurtigt udfolde og blive chaperone-aktive under specifikke stress betingelser. Når den er aktiveret, disse konditionelt uordnede chaperoner binde til et stort antal forskellige aggregering-tilbøjelige proteiner, forhindre deres aggregering og enten direkte eller indirekte fremmer protein refoldning ved tilbagevenden til ikke-stressbetingelser. Den primære metode til at få en mere detaljeret forståelse om mekanismen for deres aktivering og klient anerkendelse indebærer rensning og subsequent karakterisering af disse proteiner under anvendelse af in vitro chaperone assays. Opfølgning in vivo stress analyser er helt afgørende for selvstændigt bekræfte opnåede in vitro resultater. Denne protokol beskriver in vitro og in vivo-metoder til at karakterisere chaperonaktivitet af E. coli HdeB, en syre-aktiveret chaperone. Lysspredningsmålinger blev anvendt som en bekvem udlæsning for HdeB kapacitet til at forebygge syre-induceret aggregering af en etableret model klient protein, MDH, in vitro. Analytiske ultracentrifugering eksperimenter blev anvendt til at afsløre kompleksdannelse mellem HdeB og dets klient protein LDH, at kaste lys ind i skæbne klient proteiner når de vender tilbage til ikke-stress betingelser. Enzymatisk aktivitet assays af klienten proteiner blev udført for at overvåge virkningerne af HdeB på pH-induceret klient inaktivering og genaktivering. Endelig blev overlevelsesstudier anvendes til monitor indflydelse af HdeB s chaperone funktion in vivo.

HdeA og HdeB er homologe E. coli proteiner, kendt for at beskytte periplasmatiske proteiner mod syre stresstilstande 10. Vores arbejde viste, at ligner HdeA, HdeB fungerer også som en syre aktiveret molekylær chaperone. Men i modsætning til HdeA, HdeB funktioner ved et pH, der stadig potentielt bakteriedræbende, men signifikant højere end pH-optimum på HdeA 6,9,10,22. For at undersøge pH-optimum på HdeB s chaperone-aktivitet in vitro, blev ...

Watch this Scientific Journal Video about Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo at JoVE.com

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Denne undersøgelse beskriver biofysiske, biokemiske og molekylære teknikker til at karakterisere chaperonaktivitet af Escherichia coli HdeB under sure pH-betingelser. Disse metoder er blevet anvendt med succes til andre syre-beskyttende chaperoner såsom HdeA og kan ændres til at arbejde for andre chaperoner og stresstilstande.

Påvisning af pH-afhængige aktivitet af<em&gt; Escherichia coli</em&gt; Chaperone HdeB<em&gt; In vitro</em&gt; og<em&gt; In vivo</em

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

Research

JoVE Journal

Biochemistry

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

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

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

Instrueret Protein Emballage inden ydermembranvesikler fra<em&gt; Escherichia coli</em&gt;: Design, produktion og oprensning

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Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

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Rab10 fosforylering påvisning af LRRK2 aktivitet ved hjælp af SDS-PAGE med en fosfat-bindende Tag

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of ...

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Quantification of Hypopigmentation Activity In Vitro

Kvantificering af hypopigmentering aktivitet In Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

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With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts ...

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

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Site-Directed mutagenese for In Vitro og i Vivo eksperimenter eksemplificeret med RNA interaktioner i Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic ...

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

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Site Specifik lysin acetylering af Histoner for nukleosom rekonstitution ved hjælp af genetisk kode ekspansion i Escherichia coli

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Extraction and Visualization of Protein Aggregates after Treatment of Escherichia coli with a Proteotoxic Stressor

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The exposure of living organisms to environmental and cellular stresses often causes disruptions in protein homeostasis and can result in protein ...