Name: expression and purification of virus-like particles for vaccination
Uploaded: 2016-06-02T15:00:00
Description: Watch this Scientific Journal Video about Expression and Purification of Virus-like Particles for Vaccination at JoVE.com

Vi vill erkänna tidigare medlemmar av Ross lab som har bidragit till att optimera protokollet för maximal effektivitet och avkastning.

1. Mammalian Expression System för generering av influensa H3N2 VLP <ol><li> Subklon viral strukturella glykoproteiner hemagglutinin (HA), neuraminidas (NA), och humant immunbristvirus (HIV) Gag p55 i eukaryota expressionsvektorer, såsom tidigare beskrivits pTR600. 7 </li><li> Amplifiera DNA i kemiskt kompetenta Escherichia coli (t.ex., DH5a) och isolera transfektion-grade plasmid med användning av en plasmid-reningskit enligt tillverkarens instruktioner. Mängden DNA är beroende av avkastninge...

<ol>
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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=Viruslike+particle+vaccine+induces+protection+against+respiratory+syncytial+virus+infection+in+mice.">Viruslike particle vaccine induces protection against respiratory syncytial virus infection in mice.</a> J Infect Dis. 204, 987-995 (2011).</li><li>Ross, T. M., Tang, X., Lu, H. R., Olagnier, D., Kirchenbaum, G. A., Evans, J. 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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=Balanced+Hemagglutinin+and+Neuraminidase+Activities+Are+Critical+for+Efficient+Replication+of+Influenza+A+Virus.">Balanced Hemagglutinin and Neuraminidase Activities Are Critical for Efficient Replication of Influenza A Virus.</a> J Virol. 74, 6015-6020 (2000).</li><li>Jarvis, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Enzymology. 463, 191-222 (2009).</li><li>Pijlman, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enveloped+virus-like+particles+as+vaccines+against+pathogenic+arboviruses.">Enveloped virus-like particles as vaccines against pathogenic arboviruses.</a> Biotechnol J. 10, 659-670 (2015).</li><li>Park, J. K., 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=Protective+efficacy+of+crude+virus-like+particle+vaccine+against+HPAI+H5N1+in+chickens+and+its+application+on+DIVA+strategy.">Protective efficacy of crude virus-like particle vaccine against HPAI H5N1 in chickens and its application on DIVA strategy.</a> Influenza Other Respir Viruses. 7, 340-348 (2013).</li><li>Gangadharan, D., Smith, J., Weyant, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosafety+Recommendations+for+Work+with+Influenza+Viruses+Containing+a+Hemagglutinin+from+the+A/goose/Guangdong/1/96+Lineage.+MMWR.+Recommendations+and+reports+:+Morbidity+and+mortality+weekly+report.">Biosafety Recommendations for Work with Influenza Viruses Containing a Hemagglutinin from the A/goose/Guangdong/1/96 Lineage. MMWR. Recommendations and reports : Morbidity and mortality weekly report.</a> Recommendations and reports / Centers for Disease Control. 62, 1-7 (2013).</li></ol>

Vi har använt den teknik som beskrivs ovan för att framgångsrikt uttrycka och rena direktörerna och VLP sammansatta av flera strukturella proteiner för olika patogener. I allmänhet använder vi däggdjursexpressionssystem för att generera våra VLP. Men i våra händer, som härrör från däggdjur-cell Chik VLP avkastningen var låg. CHIK VLP Utbytet var mer robust när man använder ett rekombinant baculovirus-insektscellsystem. I allmänhet, baculovirus-insektscellsystem ger större mängder av rekombinanta pr...

Virusliknande partiklar (VLP) är en godkänd teknik för mänsklig vaccination. Faktum är att vissa av de mer contemporarily licensierade vacciner, inklusive humant papillomvirus (HPV) och hepatit Β (HepΒ) vacciner använder detta tillvägagångssätt. VLP bildas av strukturproteiner kan självorganisering. De monterade partiklarna härma virus morfologier, men kan inte infektera eller replikera eftersom de saknar virala genom. VLP kan uttryckas och renas från ett antal prokaryota och eukaryota system. En genomgång av litteraturen visar att olika uttryckssystem används på följande priser: bakterier - 28%, jäst - 20%, växt - 9%, insekt - 28%, och däggdjurs - 15% 1. Notera är HPV-vacciner baserade på L1 kapsidproteinet produceras i jäst (Gardasil) eller i ett insektscellsystem (Cervarix) 2. HepΒ vacciner, Recombivax och Engerix-Β, är också produceras i jäst, och är sammansatta av HepΒ ytantigen 3, 4. Vi använder däggdjurs- och insektscellexpressionssystem för att producera VLP som kräver samexpression av multipla strukturella proteiner för montering. Vårt arbete är inriktat på design, produktion och rening av VLP-baserade vacciner mot humana patogener: influensavirus, respiratoriskt syncytialvirus (RSV), denguevirus (DENV), och chikungunyaen virus (CHIKV). Våra metoder är tillräckligt flexibla för att medge samuttryck av de multipla strukturproteiner från flera expressionsplasmider, eller en enda expressionsplasmid (Figur 1). Tidigare har vi producerat och renat H5N1 VLP sammansatta från samexpression av plasmider som kodar influensa hemagglutinin (HA), neuraminidas (NA), och matrisen (M1) i humana embryonala njur 293T-celler 5,6. Generna kodon-optimerad för uttryck i däggdjursceller och klonades i pTR600, en eukaryot expressionsvektor innehållande de cytomegalovirus omedelbar-tidig promotor plus intron A för att initiera transcription av eukaryota skär och bovint tillväxthormon polyadenyleringssignal för terminering av transkription 7. Ett liknande tillvägagångssätt med hjälp av tre-plasmiden samexpression av HA, NA (figur 1A) och en alternativ viral matrisprotein, HIV Gag p55, användes för generering av mänskligt säsongsinfluensa subtyp H3N2 VLP i denna studie och har visat sig generera VLP av liknande storlek som partiklar influensa 8. Även influensavacciner övervägande framkalla anti-HA-antikroppar, tillsättning av influensa neuraminidas förmedlar sialidasaktivitet att möjliggöra VLP spirande från transfekterade celler 9 och även medföra ytterligare immunogena mål. För att producera RSV VLP, vi valde också den obesläktade kärnan av HIV Gag att utforma prototypiska vacciner som uppvisar uteslutande RSV ytglykoproteiner att ytterligare demonstrera flexibiliteten hos VLP bildning med användning av HIV Gag, såsom tidigare beskrivits, och som kännetecknas av elektronmikroskopi 6,10. andrahar tidigare visat att VLP presenterar RSV glykoproteiner kan monteras med hjälp av olika virala komponenter från Newcastlesjuka-virus (NDV) 11, och influensa matris 12. Fullängds ytglykoprotein sekvenser användes i denna studie att behålla nativa konformationer som kan vara nödvändiga för funktionell receptorbindning och antikroppsanalys erkännande genom enzymkopplad immunosorbentanalys (ELISA). Våra exempel för användning av enstaka plasmid expressionssystem för att generera partiklar är DENV och CHIKV. I fallet med DENV, kan vi producera subvirala partiklar (direktörerna) med ingen kapsid i 293T-celler från en enda plasmid innehållande en prM / E-strukturgenen expressionskassett 13. Termen SVP används för att beteckna att det saknas en kärna eller kapsidprotein vid montering av virala strukturella proteiner. CHIK VLP kan också framställas med användning av en enda plasmid innehållande en CHIKV strukturell gen kassett, som kodar för kapsid-och höljesproteiner, eller i insect celler genom infektering med ett rekombinant baculovirus som kodar för samma strukturella genkassetten optimerad för insektscelluttryck (Figur 1B - C). Slutresultatet av uttrycket tillvägagångssätt diskuteras ovan är frisättningen av VLP in i cellodlingsmedium som sedan kan renas via ultracentrifugering genom en glycerolkudde 20%. I denna rapport presenterar vi metoder för att uttrycka och rena dessa VLP från däggdjurs- och insektscellsystem.

<table border="0" cellpadding="0" cellspacing="0" width="786">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Plasmid Maxi Kit</td>
			<td style="width:167px;">Qiagen</td>
			<td style="width:119px;">12163</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">DMEM</td>
			<td style="width:167px;">Cellgro</td>
			<td>10-013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Lipofectamine</td>
			<td style="width:167px;">Life Technologies</td>
			<td>L3000075</td>
			<td>Lipofectamine 2000, Lipofectamine 3000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Opti-MEM I</td>
			<td style="width:167px;">Life Technologies</td>
			<td>31985062</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Bac-to-Bac Baculovirus Expression System</td>
			<td style="width:167px;">Life Technologies</td>
			<td style="width:119px;">10359-016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Cimarec I 6 multipoint stirrer plate</td>
			<td style="width:167px;">ThermoFisher Scientific</td>
			<td style="width:119px;">50094596</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Polyclear ultracentrifuge tubes</td>
			<td style="width:167px;">Seton</td>
			<td style="width:119px;">7025</td>
			<td>Confirm appropriate tubes for ultracentrifuge and bucket size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Micro BCA Protein Assay Kit</td>
			<td style="width:167px;">ThermoFisher Scientific</td>
			<td style="width:119px;">23235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Phosphate citrate buffer</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:119px;">P4922</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">o-phenylenediamine dihydrochloride&#160;</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:119px;">P8287</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">0.22 &#956;m vacuum filter top (500 mL)</td>
			<td style="width:167px;">Nalgene</td>
			<td style="width:119px;">569-0020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Glycerol</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:119px;">G5516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">H2SO4</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:119px;">339741&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Sf-900 II SFM</td>
			<td style="width:167px;">ThermoFisher Scientific</td>
			<td style="width:119px;">10902-096</td>
			<td></td>
		</tr>
	</tbody>
</table>

expression and purification of virus-like particles for vaccination

Virusliknande partiklar (VLP) och subvirala partiklar (direktörerna) är ett alternativ till viralt vaccin design som erbjuder fördelarna med ökad biosäkerhet och stabilitet över användningen av levande patogener. Icke-infektiös och självsamlande, VLP används för att presentera strukturella proteiner som immunogener, förbi behovet av levande patogener eller rekombinanta virusvektorer för antigenleverans. I den här artikeln visar vi de olika stadierna av VLP design och utveckling för framtida tillämpningar inom preklinisk djurförsök. Förfarandet innefattar följande steg: val av antigen, uttryck av antigen i cellinje val, rening av VLP / direktörerna och kvantifiering för antigen dosering. Vi visar användning av både däggdjurs- och insektscellinjer för uttryckning av våra antigener och visa hur metoder skiljer sig i utbyte. Den metod som presenteras kan tillämpas på en mängd olika patogener och kan åstadkommas genom att ersätta antigenerna med immunogen structural proteiner av användarens mikroorganismen av intresse. VLP och direktörerna hjälpa till med antigen karakterisering och urval av de bästa vaccinkandidater.

VLP avkastningen var variabel genom virusantigen konstruktion design. I detta protokoll har vi visat användning av insekts- och däggdjursceller för produktion av SVP eller VLP i supernatanten och rening genom ultracentrifugering. Fyra subtyper av DENV prM / E strukturella genuttryck kassetter användes för att konstruera de versioner av DENV direktörerna (avgränsade som 1-4) i tabell 1 och visar ett område mellan 1,1 till 2,6 mg totalt protein i 0,6 ml volymer. F?...

Watch this Scientific Journal Video about Expression and Purification of Virus-like Particles for Vaccination at JoVE.com

Expression and Purification of Virus-like Particles for Vaccination

Här presenterar vi ett protokoll för syntes av virusliknande partiklar med hjälp av antingen baculovirus eller däggdjursexpressionssystem, och ultracentrifuge rening. Detta mycket anpassningsbar metod används för att identifiera virala antigener som vaccin mål på ett säkert och flexibelt sätt.

Expression och rening av virusliknande partiklar för Vaccination

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased ...

The overall goal of this procedure is to purify virus-like particles, or VLPs, expressed and secreted by mammalian or insect-cell expression systems. This method can answer key questions in a vaccinoly's field, such as how to express and purify conformationally relevant viral antigens in a safe and flexible manner. The main advantage of this technique is that it does not require protein precipitation using polyethylene glycol or preparation of multiple density layers to concentrate VLPs.Though this method can provide insight into the identification of viral antigens as vaccine targets, VLPs can also be used as tools for disease diagnosis and serology. Visual demonstration of this method is critical as a glycerol underlay in VLP resuspension steps are difficult to learn because beginners may fail to exercise proper technique. On the day of transvection prepare liposome and DNA solutions per the manufacturer's recommendations.Transvect the DNA cells with a DNA composition of one to one to two of HA to NA to Gag and a total DNA quantity of 40 micrograms per T150 flask. Repeat this step to create nine T150 flasks, for a total volume of 200 milliliters. Next, dilute the DNA and liposome solutions in serum-free transvection media without antibiotics, so that each flask contains a total volume of 24 milliliters.Return the flasks to the incubator and maintain the cells and transvection culture medium until the day a virus-like particle, or VLP, harvest. Transfer the supernatant into 50 milliliter conical tubes to harvest the culture from the cells after 72 to 96 hours post-transvection. Spin the cells down to pellet the cellular debris.Collect the supernatants and filter them through a 0.22 micron pour membrane. Culture previously prepared spodoptera frugiperda, or Sf9 cells, in suspension in spinner flasks, by continuously stirring at 130 rpm on a multi-point stirrer plate system. Maintain culture volumes at no more than half the volume of the spinner flask for proper aeration.Next, express CHIK VLPs by infecting 250 milliliters of Sf9 cells in a spinner flask at a density of two times ten to the six cells per milliliter with previously prepared recombinant baculovirus, and return the cells to a 28 degree Celsius incubator. Use trypan blue exclusion to determine if the cell viability has decreased to 70 to 80%Upon confirmation, transfer the cultures directly from suspension into 50 milliliter conical tubes and spin the cells down. Collect the supernatants and filter them through a 0.22 micron pour membrane before sedimentation.Sterilize six 25 millimeter by 89 millimeter open top ultracentrifuge tubes with 70%ethanol. Then, ensure the ethanol has dried off completely. Load 32 milliliters of supernatants into the clean tubes.Next, carefully underlay supernatants with three milliliters of sterile 20%glycerol and PBS. Underlay the glycerol slowly to avoid disruption of the thin density layer between the glycerol and the VLP solution. Dispensing the glycerol too quickly will cause the glycerol and the VLP solutions to mix, reducing VLP sedimentation.Make sure the tubes are balanced, then start the spin. After four hours, remove the tubes from the centrifuge. Aspirate the supernatant, being careful not to dislodge the pellet from the tube.Resuspend the sedimented VLPs in at least 100 microliters at the bottom of the tubes with sterile PBS by gently and slowly pipetting up and down. Resuspension of the VLP pellet should be performed with gentle, slow repeated aspiration and expulsion of PBS using a pipette. Avoid the creation of bubbles to limit VLP loss.Finally, store the resuspended VLPs. VLP volumes and total protein yield from a conventional BCA assay were obtained from a variety of SVP and VLP constructs. CHIK SVP yields from Sf9 cells range between 0.008 to 0.016 milligrams of total protein per milliliter of supernatant volume.While production through mammalian 293 T-cells yields a ten-fold reduction of protein. This electron micrograph image shows that HA Gag core influenza VLPs are successfully expressed, assembled and purified as VLPs. Well attempting this procedure it's important to remember to transvect and infect only healthy and viable cell cultures as this will effect overall protein expression levels.Following this procedure, other methods like BCA, ELIZA, and HA assays can be performed in order to measure total protein content, specific antigen content and expression of the functional HA.Don't forget that working with an ultracentrifuge can be dangerous and precautions such as balancing the tubes, using only recommended rotors and speeds, and proper supervision of new lab personnel should always be taken while performing this procedure.

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Research

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Biology

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Beredningen av primära hematopoietisk cell kulturer från Murine Bone Marrow för elektroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Färgning av proteinerna i geler med Coomassie G-250 utan organiska lösningsmedel och ättiksyra

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Använda Gene Pulser MXcell elektroporation System till transfektera primärceller med hög verkningsgrad

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

Hjälp av ett automatiserat celltalsräknare att förenkla Studier Gene Expression: siRNA Knockdown av IL-4 beroende genuttryck i Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated anti-cancer drugs or antibiotics, is a current serious problem in public health. This multidrug resistance is largely due to energy-dependent drug efflux pumps. The pumps expel anti-cancer drugs or antibiotics into the external medium, lowering their intracellular concentration below a toxic threshold. We are studying multidrug resistance in Pseudomonas aeruginosa, an opportunistic bacterial pathogen that causes infections in patients with many types of injuries or illness, for example, burns or cystic fibrosis, and also in immuno-compromised cancer, dialysis, and transplantation patients. The major MDR efflux pumps in P. aeruginosa are tripartite complexes comprised of an inner membrane proton-drug antiporter (RND), an outer membrane channel (OMF), and a periplasmic linker protein (MFP) 1-8. The RND and OMF proteins are transmembrane proteins. Transmembrane proteins make up more than 30% of all proteins and are 65% of current drug targets. The hydrophobic transmembrane domains make the proteins insoluble in aqueous buffer. Before a transmembrane protein can be purified, it is necessary to find buffer conditions containing a mild detergent that enable the protein to be solubilized as a protein detergent complex (PDC) 9-11. In this example, we use an RND protein, the P. aeruginosa MexB transmembrane transporter, to demonstrate how to express a recombinant form of a transmembrane protein, solubilize it using detergents, and then purify the protein detergent complexes. This general method can be applied to the expression, purification, and solubilization of many other recombinantly expressed membrane proteins. The protein detergent complexes can later be used for biochemical or biophysical characterization including X-ray crystal structure determination or crosslinking studies.

In this protocol we demonstrate the expression, solubilization, and purification of a recombinantly expressed membrane protein, MexB, as a soluble protein detergent complex. MexB is a multidrug resistance membrane transporter from the opportunistic bacterial pathogen Pseudomonas aeruginosa.

Expression, tvättmedel Lösningsgörande, och rening av ett membran transportören, MexB multidrogresistensgenen Protein

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

Paraffininbäddade och frysta snitt av Drosophila Vuxen Muskler

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

In vitro Beredning av Active T. castaneum Telomeras

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

Expression och rening av den cystisk fibros transmembran konduktans-regulator protein i Saccharomyces cerevisiae

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

Rening av Transkript och metaboliter från<em&gt; Drosophila</em&gt; Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Analys av global RNA syntes vid den inre cellnivå efter Hypoxi

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...