Name: enrichment of bacterial lipoproteins and preparation of n-terminal lipopeptides for structural determination by mass spectrometry
Uploaded: 2018-05-21T12:30:00
Description: Watch this Scientific Journal Video about Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry at JoVE.com

Forskning i Meredith lab blev støttet af startup midler fra Eberly College of Science (Pennsylvania State University). Vi takker Dr. Tatiana Laremore for teknisk rådgivning og adgang til udstyr på Penn State Proteomics og masse massespektrometri Core facilitet, Universitetsparken, PA, hvor massespektrometrisk analyser blev udført.

1. celle vækst og Lysis
<ol><li>Vokse bakterier i 15 mL tryptic soja bouillon (TSB) eller lignende rige medier til sene eksponentiel fase (OD600 1,0-1,5). Høste celler ved centrifugering, vaske en gang med Tris-bufferet saltvand/EDTA (TBSE) og fortsætte med protokol eller fryse indtil brug. Bemærk: TBSE: 20 mM Tris-hydrochlorid (HCl), pH 8,0, 130 mM natriumchlorid (NaCl), og 5 mM ethylendiamintetra syre (EDTA)). Lipoprotein udtryk og modifikation kan være påvirket af vækstbetingelser (fx s...

<ol>
	<li>Babu, M. M., 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=A+database+of+bacterial+lipoproteins+(DOLOP)+with+functional+assignments+to+predicted+lipoproteins.">A database of bacterial lipoproteins (DOLOP) with functional assignments to predicted lipoproteins.</a> J Bacteriol. 188 (8), 2761-2773 (2006).</li><li>Narita, S., Tokuda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+lipoproteins;+biogenesis,+sorting+and+quality+control.">Bacterial lipoproteins; biogenesis, sorting and quality control.</a> Biochim Biophys Acta. 1862 (11), 1414-1423 (2016).</li><li>Kovacs-Simon, A., Titball, R. W., Michell, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipoproteins+of+bacterial+pathogens.">Lipoproteins of bacterial pathogens.</a> Infect Immun. 79 (2), 548-561 (2011).</li><li>Nguyen, M. T., G&#246;tz, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipoproteins+of+Gram-Positive+Bacteria:+Key+Players+in+the+Immune+Response+and+Virulence.">Lipoproteins of Gram-Positive Bacteria: Key Players in the Immune Response and Virulence.</a> Microbiol Mol Biol Rev. 80 (3), 891-903 (2016).</li><li>Nakayama, H., Kurokawa, K., Lee, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipoproteins+in+bacteria:+structures+and+biosynthetic+pathways.">Lipoproteins in bacteria: structures and biosynthetic pathways.</a> FEBS J. 279 (23), 4247-4268 (2012).</li><li>Kurokawa, 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=Novel+bacterial+lipoprotein+structures+conserved+in+low-GC+content+Gram-positive+bacteria+are+recognized+by+Toll-like+receptor+2.">Novel bacterial lipoprotein structures conserved in low-GC content Gram-positive bacteria are recognized by Toll-like receptor 2.</a> J Biol Chem. 287 (16), 13170-13181 (2012).</li><li>Kurokawa, 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=The+Triacylated+ATP+Binding+Cluster+Transporter+Substrate-binding+Lipoprotein+of+Staphylococcus+aureus+Functions+as+a+Native+Ligand+for+Toll-like+Receptor+2.">The Triacylated ATP Binding Cluster Transporter Substrate-binding Lipoprotein of Staphylococcus aureus Functions as a Native Ligand for Toll-like Receptor 2.</a> J Biol Chem. 284 (13), 8406-8411 (2009).</li><li>Asanuma, M., 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=Structural+evidence+of+&#945;-aminoacylated+lipoproteins+of+Staphylococcus+aureus.">Structural evidence of &#945;-aminoacylated lipoproteins of Staphylococcus aureus.</a> FEBS J. 278 (5), 716-728 (2011).</li><li>Armbruster, K. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+extract+of+Mycoplasma+penetrans+proteinase+K-digested+lipid-associated+membrane+proteins+rapidly+activates+NF-kappaB+and+activator+protein+1.">Lipid extract of Mycoplasma penetrans proteinase K-digested lipid-associated membrane proteins rapidly activates NF-kappaB and activator protein 1.</a> Infect Immun. 67 (6), 2951-2956 (1999).</li><li>Luque-Garcia, J. L., Zhou, G., Spellman, D. S., Sun, T. -. T., Neubert, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+electroblotted+proteins+by+mass+spectrometry:+protein+identification+after+Western+blotting.">Analysis of electroblotted proteins by mass spectrometry: protein identification after Western blotting.</a> Mol Cell Proteomics. 7 (2), 308-314 (2008).</li><li>Luque-Garcia, J. L., Zhou, G., Sun, T. -. T., Neubert, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Nitrocellulose+Membranes+for+Protein+Characterization+by+Matrix-Assisted+Laser+Desorption/Ionization+Mass+Spectrometry.">Use of Nitrocellulose Membranes for Protein Characterization by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.</a> Anal Chem. 78 (14), 5102-5108 (2006).</li><li>Kurokawa, 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=Environment-mediated+accumulation+of+diacyl+lipoproteins+over+their+triacyl+counterparts+in+Staphylococcus+aureus.">Environment-mediated accumulation of diacyl lipoproteins over their triacyl counterparts in Staphylococcus aureus.</a> J Bacteriol. 194 (13), 3299-3306 (2012).</li><li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227 (5259), 680-685 (1970).</li><li>Sch&#1235;gger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tricine-SDS-PAGE.">Tricine-SDS-PAGE.</a> Nat Protoc. 1, 16-22 (2006).</li><li>Gravel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Blotting+by+the+Semidry+Method.">Protein Blotting by the Semidry Method.</a> The Protein Protocols Handbook. , 321-334 (2002).</li><li>Webster, J., Oxley, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Identification+by+Peptide+Mass+Fingerprinting+using+MALDI-TOF+Mass+Spectrometry.">Protein Identification by Peptide Mass Fingerprinting using MALDI-TOF Mass Spectrometry.</a> The Protein Protocols Handbook. , 1117-1129 (2009).</li><li>Wilkins, M. 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Protokollen heri beskriver to adskilte faser af lipoprotein karakterisering: berigelse af TX-114 fase opdeling og strukturelle fastsættelse af MALDI-TOF MS. Under TX-114 udvinding fjerner yderligere centrifugering kontaminerende proteiner, der udfældes under denne proces, efterfulgt af acetone nedbør til at give højt beriget lipoproteiner. Ved at begrænse omfanget af hvert præparat til 15 mL værd af celler, kan flere prøver let behandles parallelt, og hvis det ønskes, samles i slutningen af protokollen.
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Bakteriel lipoproteiner er karakteriseret ved en bevaret N-terminale lipid-modificerede cystein, der forankrer kugleformede protein-domæne til celle membranen overflade. De er universelt fordelt i bakterier, der udgør 2-5% af alle cellulære gener i en typisk genom1. Lipoproteiner spiller en kritisk rolle i en bred vifte af cellulære processer, herunder næringsstof optagelse, signaltransduktion, samling af proteinkomplekser, og opretholde cellens kuvert strukturelle integritet2. I patogene bakterier tjene lipoproteiner som virulens faktorer3,4. Under en infektion tilskynder anerkendelse af N-terminale lipopeptides af Toll-lignende receptor (TLR) 2 en medfødte immunrespons fjerne invaderende patogener. Afhængigt af den N-terminale acylation stat, er lipoproteiner generelt anerkendt af alternative TLR2 heterodimerisk komplekser. TLR2-TLR1 genkender N-acylated lipopeptides, mens TLR2-TLR6 bindes gratis lipopeptid α-aminosyrer termini. En gang bundet, konvergerer signaling forløbene for at fremkalde sekretion af proinflammatoriske cytokiner3,4.
Man mente tidligere, at lipoprotein fra Gram-positive bakterier var diacylated og dem fra Gram-negative bakterier blev triacylated, kun ved fravær eller tilstedeværelse af et AMID-linked fedtsyre på den bevarede N-terminale cystein rest. Denne antagelse blev understøttet af manglen sekvens orthologs i Gram-positive genomer til Lnt, Gram-negative N-acyl-transferase, der danner triacylated lipoproteiner5. Men nyere undersøgelser har afsløret lipoprotein triacylation i Gram-positive Firmicutes at manglende lnt, samt tre nye N-terminale lipoprotein strukturer, betegnes peptidyl, lyso og N -acetyl former6,7 ,8. Disse resultater rejse spørgsmål om muligt endnu-til-være-opdagede lipoprotein former, sammen med grundlæggende spørgsmål om, hvordan disse nye lipoproteiner er lavet og hvilke fysiologiske formål eller fordel forskellige former give. Desuden, de klart viser genomforskning nuværende evne til at forudsige lipoprotein struktur. Ja, vi for nylig identificeret en roman klasse af lipoprotein N-acyl transferaser, kaldet Lit, fra Enterococcus faecalis og Bacillus cereus der gør lyso-form lipoproteiner9. Dette viser, at eksperimentelt kontrollere lipoprotein struktur, som kan blive udfordrende på grund af deres ekstremt hydrofob karakter og begrænsede metoder for at karakterisere deres molekylære struktur.
For at lette undersøgelser af lipoprotein induktion af vært immunrespons, samt N-terminale Strukturbestemmelse, har vi tilpasset flere tidligere beskrevet protokoller for at rense bakteriel lipoproteiner og forberede den N-terminale tryptic lipopeptides til analyse af MALDI-TOF MS6,10,11,12. Lipoproteiner er beriget ved hjælp en etablerede Triton X-114 (herefter benævnt overfladeaktivt stof eller TX-114) fase partitionering metode, med optimering til at fjerne forurenende ikke-lipoproteiner og øge lipoprotein udbytte. Disse lipoproteiner er egnede til direkte anvendelse i TLR assays eller til yderligere rensning af SDS-PAGE. For MALDI-TOF MS, overførsel af lipoproteiner til nitrocellulose membran giver en stillads til effektiv i situ trypsin fordøjelse, vask og efterfølgende eluering fra membranen overflade, hvilket resulterer i meget renset N-terminale lipopeptides. Nitrocellulose har vist sig at lette prøve håndtering og forbedre sekvens dækning for meget hydrofobe peptider fra integreret membran proteiner13,14, samt lipoproteiner9,10 . Metoden har den yderligere fordel, fractionating peptider baseret på polaritet, så at mellemliggende vask løsninger kan analyseres for høj genkendelsessikkerhed protein identifikation samtidig med N-terminalen Strukturbestemmelse i en enkelt eksperiment . Denne protokol entydigt funktioner forsætlig natrium addukt dannelse at fremme overordnede ion fragmentering mod dehydroalanyl ioner under MS/MS, medvirken i strukturelle tildeling af N- acylation stat. N-terminus er både variable og mest centrale funktion relateret til TLR anerkendelse af lipoproteiner. Tilsammen har denne protokol tilladt intensiv og reproducerbare studies på lipoproteiner, med de enkelte faser af rensning og Strukturbestemmelse af MALDI-TOF MS nemt tilpasses afhængig af det overordnede mål for eksperimentet.

<table>
	<tbody>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.01 mm Zirconia/silica beads</td>
			<td>BioSpec Products</td>
			<td>110791012</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>EMD</td>
			<td>AX0073-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>EMD</td>
			<td>AX0116-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>EMD</td>
			<td>AX0142-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Fluka Analytical</td>
			<td>09830</td>
			<td></td>
		</tr>
		<tr>
			<td>BioTrace NT Nitrocellulose</td>
			<td>PALL Life Sciences</td>
			<td>66485</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA) digest standard</td>
			<td>Protea</td>
			<td>PS-204-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Acros Organics</td>
			<td>423550025</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Fisher Scientific</td>
			<td>BP118</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC Grade water</td>
			<td>EMD</td>
			<td>WX0008-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Fisher Scientific</td>
			<td>BP535-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethyl sulfonyl fluoride (PMSF)</td>
			<td>Amresco</td>
			<td>0754</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce trypsin protease</td>
			<td>Thermo Scientific</td>
			<td>90057</td>
			<td></td>
		</tr>
		<tr>
			<td>Ponceau S</td>
			<td>Acros Organics</td>
			<td>161470100</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein LoBind Tube 0.5mL</td>
			<td>Eppendorf</td>
			<td>022431064</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>792519</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Macron Fine Chemicals</td>
			<td>7581-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid (TFA)</td>
			<td>Sigma-Aldrich</td>
			<td>299537</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-hydrochloride (HCl)</td>
			<td>Fisher Scientific</td>
			<td>BP152</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-114</td>
			<td>Sigma-Aldrich</td>
			<td>93422</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptic soy broth (TSB)</td>
			<td>BD</td>
			<td>211822</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-cyano-4-hydroxycinnamic acid (MS Grade)</td>
			<td>Sigma-Aldrich</td>
			<td>C8982</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MagNALyser</td>
			<td>Roche</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot Turbo Transfer System</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MALDI-TOF target</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultraflextreme MALDI-TOF-TOF</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td>Equipped with a 355 nm frequency-tripled Nd:YAG smartbeam-II laser</td>
		</tr>
	</tbody>
</table>

enrichment of bacterial lipoproteins and preparation of n-terminal lipopeptides for structural determination by mass spectrometry

Lipoproteiner er vigtige bestanddele af bakteriel celle kuvert og potent aktivatorer af pattedyr medfødte immunrespons. Trods deres betydning for både celle fysiologi og Immunologi, er stadig meget at blive opdaget omkring roman lipoprotein former, hvor de er syntetiseret, og effekten af de forskellige former på vært immunitet. Hvis du vil aktivere grundige undersøgelser af lipoproteiner, denne protokol beskriver en metode for bakteriel lipoprotein berigelse og forberedelse af N-terminale tryptic lipopeptides for Strukturbestemmelse af matrix assisted laser desorption ionisering-tid af en flyvning massespektrometri (MALDI-TOF MS). Udvide en etableret Triton X-114 fase partitionering metode for lipoprotein udvinding og berigelse af den bakterielle cellemembranen, protokollen indeholder yderligere trin for at fjerne ikke-lipoprotein forureninger, øge lipoprotein udbytte og renhed. Da lipoproteiner er almindeligt anvendt i Toll-lignende receptor (TLR) assays, det er afgørende at først karakterisere den N-terminale struktur af MALDI-TOF MS. Herein, en metode er præsenteret for at isolere koncentrerede hydrofobiske peptider beriget med N-terminalen lipopeptides egnet til direkte analyse af MALDI-TOF MS/MS. lipoproteiner, der har været adskilt af Sodium Dodecyl sulfat Poly-acrylamid gelelektroforese (SDS-PAGE) overføres til en nitrocellulose membran, fordøjet i situ med Trypsin, vaskes sekventielt for at fjerne polære tryptic peptider, og endelig elueret med chloroform-methanol. Når kombineret med MS i de mere polære trypsinized peptider fra vask løsninger, giver denne metode mulighed for at identificere lipoprotein såvel karakterisere sin N-terminus i et enkelt eksperiment. Forsætlig natrium addukt dannelse kan også anvendes som et redskab til at fremme mere strukturelt informative fragmentering spektre. I sidste ende, berigelse af lipoproteiner og bestemmelse af deres N-terminale strukturer vil muliggøre mere omfattende undersøgelser på denne allestedsnærværende klasse af bakterielle proteiner.

En skematisk af protokollen er fastsat i figur 1. Lipoprotein-beriget brøkdel udvundet af Enterococcus faecalis ATCC 19433 af TX-114 er vist i figur 2. Til sammenligning, er den banding mønster af de bundfaldne proteinfraktion også vist. Proteiner fra denne fraktion blev bekræftet af MALDI-MS til at være meget rigelige, kontaminerende proteiner end lipoproteiner (tabel 1). Massespektre i <strong cla...

Watch this Scientific Journal Video about Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry at JoVE.com

Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry

Berigelse af bakteriel lipoproteiner og forberedelse af N-terminale Lipopeptides for Strukturbestemmelse af massespektrometri

Berigelse af bakteriel lipoproteiner ved hjælp af en nonionisk overfladeaktivt stof fase partitionering metode er beskrevet til direkte brug i TLR assays eller andre programmer. Yderligere skridt er detaljeret at forberede N-terminale tryptic lipopeptides for strukturel karakterisering af massespektrometri.

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their ...

Research

JoVE Journal

Immunology and Infection

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Spyt, spytkirtel, og hæmolymfe Collection fra Ixodes scapularis Flåter

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

Opdagelsen af ​​nye intracellulære patogener af Amoebal cokultur og Amoebal Enrichment Approaches

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

Rapid Identifikation af Gram-negative bakterier fra Blood kulturvæsken Brug MALDI-TOF-massespektrometri

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Fremstilling af en blodkultur Pellet til hurtig bakteriel identifikation og antibiotikaresistens undersøgelserne

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

Electroporation of Functional Bacterial Effectors into Mammalian Cells

Elektroporation af funktionelle Bakterielle effektorer i pattedyrceller

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Generation og Multi-fænotypisk High-indhold Screening af<em&gt; Coxiella burnetii</em&gt; Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Alternative In Vitro metoder til bestemmelse af Viral kapsid strukturelle integritet

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...

Identification of Nucleolar Factors During HIV-1 Replication Through Rev Immunoprecipitation and Mass Spectrometry

Identifikation af Nukleolarfaktorer under HIV-1-replikation gennem rev-Immunopræcipitation og massespektrometri

The HIV-1 infectious cycle requires viral protein interactions with host factors to facilitate viral replication, packaging, and release. The infectious ...