Name: identifying amino acid overproducers using rare-codon-rich markers
Uploaded: 2019-06-24T13:30:00
Description: Watch this Scientific Journal Video about Identifying Amino Acid Overproducers Using Rare-Codon-Rich Markers at JoVE.com

Arbejdet blev understøttet af National Natural Science Foundation i Kina (Grant No. 21676026), det nationale Key R & D program i Kina (Grant No. 2017YFD0201400), og China postdoc Science Foundation (Grant No. 2017M620643). Værker på UCLA Institute of avancement (Suzhou) blev støttet af de interne tilskud fra Jiangsu-provinsen og Suzhou Industrial Park.

1. konstruktion af plasmider udtrykker de sjældne-codon-rige markørgener
<ol><li>Vælg en markør gen, der indeholder et passende antal af de fælles kodon for den målrettede aminosyre. Bemærk: for L-leucin anvendes kanamycin-resistens genet kanR, som indeholder 29 leucin codons, hvoraf 27 er almindeligt codons, til opførelse af udvælgelsessystemet13. Gfp -genet, som indeholder 17 almindelige kodon ud af 19 leucin kodon, eller det lilla protein-kodnings-gen<...

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Antallet af sjældne kodon i markørgener og udvælgelse eller screening medium er afgørende for at hæmme protein udtryk fra de sjældne-codon-modificerede markørgener. Hvis der ikke kan påvises nogen signifikant forskel mellem protein udtryk fra Wild-type markørgener og derivater heraf, kan en forøgelse af antallet af sjældne kodon eller anvendelse af et nærings begrænset medium forstærke forskellene. Men hvis hæmnings effekten er for stærk, kan protein udtrykkene ikke inddrives, selv ved ekstra fodring af d...

Den nuværende produktion af aminosyrer er stærkt afhængig af gæring. Men, titre og udbytter for de fleste aminosyre produktion stammer er under de stigende krav fra det globale aminosyre marked, der er værd milliarder af dollars1,2. Opnåelse af højtydende aminosyre over producenter er afgørende for opgraderingen af aminosyre industrien.
Traditionel strategi til at identificere aminosyre over producenter udnytter konkurrencer mellem aminosyrer og deres analogier i proteinsyntesen3,4. Disse analogerne er i stand til at oplade tRNAs, der genkender de tilsvarende aminosyrer og dermed hæmme forlængelsen af peptidkæder, fører til anholdt vækst eller celledød5. En måde at modstå de analoge belastninger er at øge koncentrationerne af intracellulære aminosyrer. De berigede aminosyrer vil udkonkurrere analoger til finite tRNAs og sikre den korrekte syntese af funktionelle proteiner. Derfor kan stammer, der overlever analoerne vælges og er sandsynligvis over producenter af de tilsvarende aminosyrer.
Selv om det viste sig at være vellykket at udvælge over producenter for aminosyrer som L-leucin6, lider den analoge-baserede strategi af alvorlige ulemper. Et stort problem er den analoge modstand stammer fra processen med mutagenese eller gennem spontane mutationer. Stammer med resistens kan undslippe udvælgelsen ved at blokere, eksportere eller nedværdigende analoner5. En anden bekymring er de giftige bivirkninger af analoerne på andre cellulære processer7. Som en konsekvens, stammer, der overlever den analoge udvælgelse kan ikke være aminosyren over producenter, mens de ønskede over producenter kunne være falsk udryddet på grund af de negative bivirkninger.
Her præsenteres en ny strategi baseret på loven om codon bias for at opnå nøjagtige og hurtige identifikationer af aminosyre over producenter. De fleste aminosyrer er kodet af mere end en nucleotid triplet, der er begunstiget forskelligt af værts organismerne 8,9. Nogle kodon bruges sjældent i kodning sekvenser og omtales som de sjældne kodon. Deres oversættelser til aminosyrer er afhængige af de cognate tRNAs, der bærer de tilsvarende aminosyrer. Men de trnas, der genkender sjældne kodon normalt har meget lavere mængder end trnas af de fælles kodon10,11. Derfor er disse sjældne tRNAs mindre tilbøjelige til at fange de frie aminosyrer i konkurrencerne med andre isoacceptorer, og oversættelser af de sjældne-codon-rige sekvenser begynder at aftage eller endda afsluttes, når mængderne af aminosyrer er begrænset 10. oversættelserne kunne teoretisk set genoprettes, hvis der er en aminosyre overskud efter opladning af de synonyme fælles tRNAs på grund af over produktioner eller ekstra fodring af de tilsvarende aminosyrer12. Hvis det sjældne-codon-rige gen koder et valg eller screening markør, kan stammer, der udviser de tilsvarende fænotyper, så let identificeres og er sandsynligvis over producenterne af de målrettede aminosyrer.
Ovennævnte strategi anvendes til at etablere en udvælgelse og et screening system til identifikation af aminosyre over producenter. Udvælgelsessystemet bruger antibiotikaresistensgener (f. eks. kanR) som markører, mens Screenings systemet bruger de gener, som koder fluorescerende (f. eks. grønt fluorescerende protein [gfp]) eller kromogene (f. eks. prancerpurple) proteiner. Markør generne i begge systemer ændres ved at erstatte definerede numre af de fælles kodon for den målrettede aminosyre med dens synonyme sjældne alternativ. Stammer i mutation biblioteket, der havnen den sjældne-codon-rige markør genet udvælges eller screenes under ordentlige forhold, og over producenter af de målrettede aminosyrer kan let identificeres. Arbejdsprocessen begynder med opførelsen af den sjældne-codon-rige markør gensystem, efterfulgt af optimering af arbejdsvilkårene, og derefter identifikation og verifikation af aminosyren over producenter. Denne analoge-uafhængige strategi er baseret på dogmet i protein oversættelse og er praktisk talt blevet verificeret for at muliggøre præcise og hurtige identifikationer af aminosyre over producenter. Teoretisk, det kunne være direkte ansat til aminosyrer med sjældne kodon og til alle mikroorganismer. I alt vil den sjældne-codon-baserede strategi tjene som et effektivt alternativ til den konventionelle analog-baserede tilgang, når passende analogier til specifikke aminosyrer er utilgængelige, eller når en høj falsk positiv sats er den største bekymring. Nedenstående protokol bruger leucin rare codon til at demonstrere denne strategi for at identificere over producenter af Escherichia coli L-leucin.

<table>
	<tbody>
		<tr>
			<td>Acetonitrile</td>
			<td>Thermo</td>
			<td>51101</td>
			<td></td>
		</tr>
		<tr>
			<td>EasyPure HiPure Plasmid MiniPrep Kit</td>
			<td>Transgen</td>
			<td>EM111-01</td>
			<td></td>
		</tr>
		<tr>
			<td>EasyPure Quick Gel Extraction Kit</td>
			<td>Transgen</td>
			<td>EG101-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibson assembly master mix</td>
			<td>NEB</td>
			<td>E2611S</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl &#946;-D-1-thiogalactopyranoside</td>
			<td>Solarbio</td>
			<td>I8070</td>
			<td></td>
		</tr>
		<tr>
			<td>L-leucine</td>
			<td>Sigma</td>
			<td>L8000</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Biotek</td>
			<td>Synergy 2</td>
			<td></td>
		</tr>
		<tr>
			<td>n-hexane</td>
			<td>Thermo</td>
			<td>H3061</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenyl isothiocyanate</td>
			<td>Sigma</td>
			<td>P1034</td>
			<td></td>
		</tr>
		<tr>
			<td>PrancerPurple CPB-37-441</td>
			<td>ATUM</td>
			<td>CPB-37-441</td>
			<td></td>
		</tr>
		<tr>
			<td>TransStar FastPfu Fly DNA polymerase</td>
			<td>Transgen</td>
			<td>AP231-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethylamine</td>
			<td>Sigma</td>
			<td>T0886</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-high performance liquid chromatography</td>
			<td>Agilent</td>
			<td>1290 Infinity II</td>
			<td></td>
		</tr>
		<tr>
			<td>Wild type C. glutamicum</td>
			<td>ATCC</td>
			<td>13032</td>
			<td></td>
		</tr>
		<tr>
			<td>XL10-Gold E. coli competent cell</td>
			<td>Agilent</td>
			<td>200314</td>
			<td></td>
		</tr>
		<tr>
			<td>ZORBAX RRHD Eclipse Plus C18 column</td>
			<td>Agilent</td>
			<td>959759-902K</td>
			<td></td>
		</tr>
	</tbody>
</table>

identifying amino acid overproducers using rare-codon-rich markers

For at tilfredsstille det stadigt voksende marked for aminosyrer er der behov for højtydende produktions stammer. Aminosyren over producenter er konventionelt identificeret ved at udnytte de konkurrencer mellem aminosyrer og deres analoner. Men, denne analoge-baserede metode er af lav nøjagtighed, og korrekt analoner til specifikke aminosyrer er begrænset. Her præsenterer vi en alternativ strategi, der muliggør en nøjagtig, følsom og høj gennemløbs screening af aminosyre over producenter ved hjælp af sjældne-codon-rige markører. Denne strategi er inspireret af fænomenet codon skik bias i protein oversættelse, for hvilke kodon er kategoriseret i fælles eller sjældne dem baseret på deres frekvenser af forekomsten i kodning DNA. Oversættelsen af sjældne kodon afhænger af deres tilsvarende sjældne Transfer RNAs (trnas), som ikke kan oplades fuldt ud af de cognate aminosyrer under hungersnød. Teoretisk kan de sjældne tRNAs oplades, hvis der er et overskud af aminosyrer efter opladning af de synonyme fælles isoacceptorer. Derfor kan retarderede oversættelser forårsaget af sjældne kodon gendannes ved fodring eller intracellulære over produktioner af de tilsvarende aminosyrer. Under denne antagelse etableres et udvælgelses-eller screeningssystem til identificering af aminosyre overproducenter ved at erstatte de almindelige kodon af de målrettede aminosyrer med deres synonyme sjældne alternativer i antibiotikaresistensgener eller gener kodning af fluorescerende eller kromogene proteiner. Vi viser, at protein udtryk kan være stærkt hæmmet af inkorporering af sjældne kodon, og at niveauerne af proteiner korrelerer positivt med aminosyre koncentrationer. Ved hjælp af dette system, over producenter af flere aminosyrer kan let screenes ud fra mutation biblioteker. Denne sjældne-codon-baserede strategi kræver kun et enkelt modificeret gen, og værten er mindre tilbøjelige til at undslippe udvælgelsen end i andre metoder. Det tilbyder en alternativ tilgang til at opnå aminosyre over producenter.

For udvælgelsessystemet bør et kraftigt fald i OD600 for stammer, der harkedeligt det sjældne-codon-rige antibiotikaresistens-gen, observeres i forhold til den stamme, der harkedeligt det vilde antibiotikaresistens-gen, når det dyrkes i en egnet medium (figur 1a). Under de samme betingelser, faldet i celle OD600 bliver mere indlysende, da antallet af sjældne kodon i antibiotikaresistens genet stiger (figur 1a</s...

Watch this Scientific Journal Video about Identifying Amino Acid Overproducers Using Rare-Codon-Rich Markers at JoVE.com

Identifying Amino Acid Overproducers Using Rare-Codon-Rich Markers

Denne undersøgelse præsenterer en alternativ strategi for konventionel toksisk analog-baseret metode til at identificere aminosyre over producenter ved hjælp af sjældne-codon-rige markører for at opnå nøjagtighed, følsomhed, og høj gennemløb samtidigt.

Identificering af amino syre-over producenter ved hjælp af sjældne-codon-rige markører

To satisfy the ever-growing market for amino acids, high-performance production strains are needed. The amino acid overproducers are conventionally ...

Denne metode giver et effektivt alternativ til den konventionelle analog-baserede tilgang til at opnå aminosyre overproducenter. Denne teknik udkonkurrerer den traditionelle analogbaserede metode ved at give nøjagtighed, følsomhed og høj overførselshastighed samtidigt. Denne metode kaster nyt lys over forståelsen af aminosyreoverproduktionsstyrke og oversættelse af sjældne codons og kan teoretisk set anvendes på alle mikroorganismer.Denne protokol er hovedsagelig baseret på grundlæggende molekylære eksperimentelle operationer, som er nemme at følge, men kan kræve flere forsøg for at opnå et passende udvalg af belastende stringenser. En visuel demonstration af denne teknik giver en direkte forståelse af udvælgelsens ydeevne og et screeningssystem til identifikation af aminosyreproducenter. For at starte denne procedure, tilsæt vektoren i markør fragmenter, på is, i en kindtand forhold på en til et til syv punkt fem mikroliter af samleblanding til et samlet volumen på ti mikroliter.Inkubere ved 50 grader Celsius i en time. Omdan fem mikroliter af samleproduktet til 50 mikroliter af komponentceller ved 42 grader Celsius i 30 sekunder. Gendan cellerne i Super Optimal bouillon med katabolit undertrykkelse medium ved 37 grader Celsius i en time.Derefter plade dem på LB Agar medium og inkubere natten over ved 37 grader Celsius. Den næste dag, bruge en foretrukken kommerciel kit til at isolere plasmid. Først omdanne 50 mikroliter af de kompetente celler med en microliter af plasmid, der bærer wild-type KanR.Omdan et andet sæt af de kompetente celler med plasmid, der indeholder KanR RC 29 med alle leucin codoner erstattet af den sjældne codon CTA. Plade og inkuber cellekulturen som beskrevet i tekstprotokollen. Dernæst overføres de kolonier, der huser det vilde markeringsmarkørgen i det sjældne codonrige derivat individuelt, i fem milliliter femdume fortyndet LB-medium indeholdende kanamycin i en koncentration på 50 mikrogram pr. milliliter.Inkuber prøverne i en shaker ved 37 grader Celsius, mens der rystes ved 250 omdrejninger i minuttet. Derefter overføres 200 mikroliter af hver af cellekulturerne til en 96 brøndplade i tre eksemplarer på definerede tidspunkter. Brug en pladelæser til at måle OD600.Inokuler pletterne huser KanR RC 29 markør genet i to sæt af fem milliliter af 0,2 x LB medium, der indeholder kanamycin, med eller uden en levering af L-leucin. Tilføj L-leucin til et af medierne til en endelig koncentration på et gram pr. liter. Inkuber prøverne i en shaker ved 37 grader Celsius med omrystning ved 250 omdr./min., og OD600 måles for hver kultur på definerede tidspunkter.Til at begynde med omdanne 50 mikroliter af moderselskabet stamme, der anvendes til mutagenese med en mikroliter af plasmid, der bærer den vilde type GFP eller wild-type PPG. Plade og inkuber cellekulturerne som beskrevet i tekstprotokollen. Dernæst overføres kolonierne individuelt til fem milliliter af det korrekt fortyndede LB-medium.Inkuber prøverne i en shaker ved 37 grader Celsius med omrystning ved 250 omdrejninger i minuttet. For fluorescensmarkører overføres 200 mikroliter af cellekulturerne til en 96 godt klar bundplade i tre eksemplarer på definerede tidspunkter. Od600 måles i fluorescensen, og fluorescensintensiteten beregnes.For kromogene markører måles cellekulturens farveudvikling. Udfør fodring assay som tidligere beskrevet og måle fluorescens intensitet eller farveudvikling på definerede tidspunkter. Først omdannes 50 mikroliter af de mutante celler med en mikroliter af plasmid, der bærer markeringsmarkøren KanR RC 29, eller screeningsmarkørerne GFP RC eller PPG RC. Dernæst centrifuge cellekulturer på 4000 gange G i fem minutter.Ved valg kasseres supernatanten, og der tilsættes fem milliliter 0,2 x LB-medium, der indeholder kanamycin, til celler, der bærer markeringsmarkøren. Inkuber prøven natten over i en shaker ved 37 grader Celsius. Den næste dag, plade natten kultur på 0,2 x LB agar medium, der indeholder kanamycin og inkubere ved 37 grader Celsius i 12 timer.Til screening, plade det passende antal celler huser screening markør på LB agar medium, der indeholder passende antibiotika. Inkubere ved 37 grader Celsius i otte timer. Derefter vaccineres LB-mediet med det relevante antibiotikum med hver enkelt koloni fra pladen.Inkuber prøverne i en shaker ved 37 grader Celsius, mens der rystes ved 250 omdrejninger i minuttet. Mål OD600 og fluorescensen, og udregn fluorescensintensiteten, hvis der anvendes GFP RC. Mål farveudviklingen af cellekulturerne, hvis DER anvendes PPG RC.For at kontrollere aminosyren productivities af kandidaten stammer, forberede frø kultur ved podning fem milliliter LB medium med hver af de kandidat stammer og lad cellerne vokse natten over i en shaker på 250 rpm og ved 37 grader Celsius. Den næste dag, høste cellerne fra en milliliter af cellekulturen ved centrifugering ved 4000 gange G i to minutter. Kassér supernatanten og opsvar paletten igen med en milliliter sterilt vand.Inokuler 20 milliliter M9-medium, der indeholder fire procent glukose med 200 mikroliter af cellesuspensionen og inkuberes i en 250 millilitershaker ved 250 omdrejninger i minuttet og ved 37 grader Celsius i 24 timer. Den næste dag, centrifuge en milliliter af kulturmediet på 4000 gange G i fem minutter. Overfør 200 mikroliter af supernatanten til et rent 1,5 milliliterrør.Forbered L-leucin standardopløsninger ved de koncentrationer, der vises her. I en røghætte tilsættes 100 mikroliter af en millimolar triethylamin og 100 mikroliter af en molar phenyl isothiocyanat til både supernatanten og standarderne. Opløsningerne blandes forsigtigt og inkuberes ved stuetemperatur i en time.Derefter tilsættes 400 mikroliter n-hexan til samme rør og vortex det i ti sekunder. Den nederste fase indeholder aminosyrederivaterne og anvendes til HPLC-analyse. Den nederste fase filtreres gennem 0,2 mikroliterpolytetrafluorethylenmembraner.Derefter køres en mikroliter af prøven på en ultra HPLC udstyret med en C18 kolonne med en strømningshastighed på 0,42 milliliter i minuttet og ved kolonnetemperatur i 40 grader Celsius. Brug en diodearraydetektor til at detektere de målrettede aminosyrer ved 254 nanometer og beregne deres koncentrationer ved at kortlægge topområderne under standardkurven. I denne undersøgelse, aminosyre overproducenter identificeres ved hjælp af sjældne codon-rige markører til samtidig opnå nøjagtighed, følsomhed, og høj-throughput.For udvælgelsessystemet bør der observeres et kraftigt fald i OD600 for stammer, der huser det sjældne codonrige antibiotikaresistensgen, i forhold til den stamme, der huser den vilde type. Hæmning af sjældne codon på proteinudtryk for det meste finder sted under udsultede forhold. Efter ekstra fodring af den tilsvarende aminosyre, OD600 for stammen huser den sjældne codon-rige antibiotikaresistens gen stiger betydeligt.For screeningssystemet er fluorescensintensiteten i antallet af lysstofrør betydeligt lavere for den stamme, der udtrykker fluorescerende protein fra det sjældne codonrige gen end fra det vilde gen. Fodring af den tilsvarende aminosyre vil genoprette proteinudtryk fra de sjældne codon-rige gener. Når du bruger det lilla protein, er farven udviklet fra den sjældne codon-rige PPG lettere end fra det vilde gen.Det ufortyndede LB-medie giver mulighed for langsom ekspression af den sjældne codonrige PPG uden L-leucinfodring i det udtrykte lilla protein bliver synligt, når cellerne pelleteres. Dette skjuler dog ikke det faktum, at genekspression fra den sjældne codon-rige PPG forstærkes ved fodring af L-leucin. Den sjældne codon-baserede strategi kunne identificere overproducenter af de målrettede aminosyrer fra mutationsbiblioteket, og disse mutanter bør producere større mængder af de målrettede aminosyrer end forældrestammerne.Når du forsøger denne procedure, er det vigtigt at justere sjældne codon frekvens for at opnå en klar forskelsbehandling i OD eller farve mellem celler huser wild-type markører og de sjældne codon-rige derivater. Transskriptionsanalyse og genomsekvensering kunne anvendes på de udvalgte stammer for at undersøge mekanismen relateret til aminosyreproduktioner. Ved hjælp af denne teknik, aminosyre overproducenter kunne identificeres effektivt og analyse af deres genetiske oplysninger ville give ny indsigt i deres mekanismer bag aminosyre overproduktioner.Aminosyre derivatization kan være skadeligt. Sørg for at bære handsker og beskyttelsesbeklædning og udføre disse trin i en røghætte.

identifying-amino-acid-overproducers-using-rare-codon-rich-markers

Research

JoVE Journal

Biochemistry

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

Fremgangsmåde til at identificere småmolekylære inhibitorer af protein-protein-vekselvirkning mellem HCN1 og TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where real-time measurements of fluorescence and currents simultaneously report on local rearrangements and global function, respectively1. While high-resolution structural techniques such as cryo-electron microscopy or X-ray crystallography provide static images of the proteins of interest, VCF provides dynamic structural data that allows us to link the structural rearrangements (fluorescence) to dynamic functional data (electrophysiology). Until recently, the thiol-reactive chemistry used for site-directed fluorescent labeling of the proteins restricted the scope of the approach because all accessible cysteines, including endogenous ones, will be labeled. It was thus required to construct proteins free of endogenous cysteines. Labeling was also restricted to sites accessible from the extracellular side. This changed with the use of Fluorescent Unnatural Amino Acids (fUAA) to specifically incorporate a small fluorescent probe in response to stop codon suppression using an orthogonal tRNA and tRNA synthetase pair2. The VCF improvement only requires a two-step injection procedure of DNA injection (tRNA/synthetase pair) followed by RNA/fUAA co-injection. Now, labelling both intracellular and buried sites is possible, and the use of VCF has expanded significantly. The VCF technique thereby becomes attractive for studying a wide range of proteins and &#8211; more importantly &#8211; allows investigating numerous cytosolic regulatory mechanisms.

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

This article describes an enhancement of conventional Voltage-Clamp Fluorometry (VCF) where Fluorescent Unnatural Amino Acids (fUAA) are used instead of maleimide dyes, to probe structural rearrangements in ion channels. The procedure includes Xenopus oocyte DNA injection, RNA/fUAA coinjection, and simultaneous current and fluorescence measurements.

Spændingsklemme Fluorometri i<em&gt; Xenopus</em&gt; Oocytter ved hjælp af fluorescerende unaturlige aminosyrer

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where ...

Hydrolysis of a Ni-Schiff-Base Complex Using Conditions Suitable for Retention of Acid-labile Protecting Groups

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide sequences. Synthesis of some unnatural amino acids often includes the use of a precursor consisting of a Schiff-base stabilized by a nickel cation. Unnatural side-chains can be installed on an amino acid backbone found in this Schiff-base complex. The resulting unnatural amino acid can then be isolated from this complex using hydrolysis of the Schiff-base, typically by employing reflux in strongly acidic solution. These highly acidic conditions may remove acid-labile side-chain protecting groups necessary for the unnatural amino acids to be used in microwave-assisted solid-phase peptide synthesis. In this work, we present an efficient hydrolysis and subsequent Fmoc protection of an amino acid isolated from a Ni-Schiff base complex. Hydrolysis conditions presented in this work are suitable for retention of acid-labile side-chain protecting groups and may be adaptable to a variety of unnatural amino acid substrates.

Here, we present an efficient hydrolysis and subsequent Fmoc protection of an amino acid isolated from a Ni-Schiff-base complex. Hydrolysis conditions presented here are suitable for use when retention of acid-labile side-chain protecting groups is required. This technique may be adaptable to a variety of unnatural amino acid substrates.

Hydrolyseprodukter af en Ni-Schiff-Base Complex Brug af betingelser egnet til Bevarelse af syrelabile Protecting Groups

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide ...

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. Nucleases display a variety of vital physiological roles in prokaryotic and eukaryotic organisms, ranging from maintaining genome stability to providing protection against pathogens. Altered nuclease activity has been associated with several pathological conditions including bacterial infections and cancer. To this end, nuclease activity has shown great potential to be exploited as a specific biomarker. However, a robust and reproducible screening method based on this activity remains highly desirable.
Herein, we introduce a method that enables screening for nuclease activity using nucleic acid probes as substrates, with the scope of differentiating between pathological and healthy conditions. This method offers the possibility of designing new probe libraries, with increasing specificity, in an iterative manner. Thus, multiple rounds of screening are necessary to refine the probes&#39; design with enhanced features, taking advantage of the availability of chemically modified nucleic acids. The considerable potential of the proposed technology lies in its flexibility, high reproducibility, and versatility for the screening of nuclease activity associated with disease conditions. It is expected that this technology will allow the development of promising diagnostic tools with a great potential in the clinic.

Altered nuclease activity has been associated with different human conditions, underlying its potential as a biomarker. The modular and easy to implement screening methodology presented in this paper allows the selection of specific nucleic acid probes for harnessing nuclease activity as a biomarker of disease.

Kinetisk screening af Nukleaseaktivitet ved hjælp af nukleinsyre sonder

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...

Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. Post-translational modifications (PTMs) of histones, which are highly dynamic and can be added or removed by enzymes, play critical roles in regulating gene expression. In plants, epigenetic factors, including histone PTMs, are related to their adaptive responses to the environment. Understanding the molecular mechanisms of epigenetic control can bring unprecedented opportunities for innovative bioengineering solutions. Herein, we describe a protocol to isolate the nuclei and purify histones from sorghum leaf tissue. The extracted histones can be analyzed in their intact forms by top-down mass spectrometry (MS) coupled with online reversed-phase (RP) liquid chromatography (LC). Combinations and stoichiometry of multiple PTMs on the same histone proteoform can be readily identified. In addition, histone tail clipping can be detected using the top-down LC-MS workflow, thus, yielding the global PTM profile of core histones (H4, H2A, H2B, H3). We have applied this protocol previously to profile histone PTMs from sorghum leaf tissue collected from a large-scale field study, aimed at identifying epigenetic markers of drought resistance. The protocol could potentially be adapted and optimized for chromatin immunoprecipitation-sequencing (ChIP-seq), or for studying histone PTMs in similar plants.

The protocol has been developed to effectively extract intact histones from sorghum leaf materials for profiling of histone post-translational modifications that can serve as potential epigenetic markers to aid engineering drought resistant crops.

Isolering af Histone fra Sorghum Leaf Tissue for Top Down Massespektrometri Profilering af potentielle epigenetiske markører

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. ...

Estimation of Plant Biomass Lignin Content using Thioglycolic Acid (TGA)

Lignin is a natural polymer that is the second most abundant polymer on Earth after cellulose. Lignin is mainly deposited in plant secondary cell walls and is an aromatic heteropolymer primarily composed of three monolignols with significant industrial importance. Lignin plays an important role in plant growth and development, protects&#160;from biotic and abiotic stresses, and in the quality of animal fodder, the wood, and industrial lignin products. Accurate estimation of lignin content is essential for both fundamental understanding of the lignin biosynthesis and industrial applications of biomass. The thioglycolic acid (TGA) method is a highly reliable method of estimating the total lignin content in the plant biomass. This method estimates the lignin content by forming thioethers with the benzyl alcohol groups of lignin, which are soluble in alkaline conditions and insoluble in acidic conditions. The total lignin content is estimated using a standard curve generated from commercial bamboo lignin.

Estimation of Plant Biomass Lignin Content using Thioglycolic Acid TGA

Here, we present a modified TGA method for estimation of lignin content in herbaceous plant biomass. This method estimates the lignin content by forming specific thioether bonds with lignin and presents an advantage over the Klason method, as it requires a relatively small sample for lignin content estimation.

Skøn over indholdet af plantebiomasse lignin ved hjælp af thioglykolsyre (TGA)

Lignin is a natural polymer that is the second most abundant polymer on Earth after cellulose. Lignin is mainly deposited in plant secondary cell walls ...

Optimized Incorporation of Alkynyl Fatty Acid Analogs for the Detection of Fatty Acylated Proteins using Click Chemistry

Fatty acylation, the covalent addition of saturated fatty acids to protein substrates, is important in regulating a myriad of cellular functions in addition to its implications in cancer and neurodegenerative diseases. Recent developments in fatty acylation detection methods have enabled efficient and non-hazardous detection of fatty acylated proteins, particularly through the use of click chemistry with bio-orthogonal labeling. However, click chemistry detection can be limited by the poor solubility and potential toxic effects of adding long chain fatty acids to cell culture. Described here is a labeling approach with optimized delivery using saponified fatty acids in combination with fatty-acid free BSA, as well as delipidated media, which can improve detection of hard to detect fatty acylated proteins. This effect was most pronounced with the alkynyl-stearate analog, 17-ODYA, which has been the most commonly used fatty acid analog in click chemistry detection of acylated proteins. This modification will improve cellular incorporation and increase sensitivity to acylated protein detection. In addition, this approach can be applied in a variety of cell types and combined with other assays such as pulse-chase analysis, stable isotope labeling with amino acids in cell culture, and mass spectrometry for quantitative profiling of fatty acylated proteins.

The study of fatty acylation has important implications in cellular protein interactions and diseases. Presented here is a modified protocol to improve click chemistry detection of fatty acylated proteins, which can be applied in various cell types and combined with other assays, including pulse-chase and mass spectrometry.

Optimeret inkorporering af alkynylfedtsyreanaloger til påvisning af fedtaylerede proteiner ved hjælp af klikkemi

Fatty acylation, the covalent addition of saturated fatty acids to protein substrates, is important in regulating a myriad of cellular functions in ...

Folding and Probing of Nucleic Acid Constructs

Begin by pipetting G4-TBA, single-stranded TBA, and double-stranded TBA solutions into three different tubes for folding. Heat the samples to 95 degrees Celsius for five minutes. Let them cool slowly to room temperature.Once the tubes have cooled to room temperature, centrifuge the tubes. Next, organize three sets each of seven empty autoclaved 0.5-milliliter tubes. Add three microliters of ultrapure water into each tube.Pipette five microliters of the folded nucleic acid constructs into each tube of the set. Next, add two microliters of either 25 or 250-micromolar B-CEP1 to obtain the final probe concentration of five and 50 micromolar respectively. To the three control tubes, add ultrapure water instead of the compound and incubate all the samples at 37 degrees Celsius.Stop the reaction by placing the tubes at 20 degrees Celsius, then dry the samples in a vacuum centrifuge.

Foldning og sondering af nukleinsyrekonstruktioner

<meta charset="utf8"></head><body>Forudsigelse af aminosyrepræferencer for protein-protein-bindingsgrænseflader

Computational Prediction of Amino Acid Preferences of Potentially Multispecific Peptide-Binding Domains Involved in Protein-Protein Interactions

Many protein-protein interactions involve the binding of short protein segments to peptide-binding domains. Usually, such interactions require the recognition of linear motifs with variable conservation. The combination of highly conserved and more variable regions in the same ligands often contributes to the multispecificity of binding, a common property of enzymes and cell signaling proteins. Characterization of amino acid preferences of peptide-binding domains is important for the design of mediators of protein-protein interactions (PPIs). Computational methods are an efficient alternative to the often costly and cumbersome experimental techniques, enabling the design of potential mediators that can be later validated in downstream experiments. Here, we described a methodology using the Pepspec application of the Rosetta molecular modeling package to predict the amino acid preferences of peptide-binding domains. This methodology is useful when the structure of the receptor protein and the nature of the peptide ligand are both known or can be inferred. The methodology starts with a well-characterized anchor from the ligand, which is extended by randomly adding amino acid residues. The binding affinity of peptides generated this way is then evaluated by flexible-backbone peptide docking in order to select the peptides with the best predicted binding scores. These peptides are then used to calculate amino acid preferences and to optionally compute a position-weight matrix (PWM) that can be used in further studies. To illustrate the application of this methodology, we used the interaction between subunits of human interferon regulatory factor 5 (IRF5), previously known to be multispecific but globally guided by a short conserved motif called pLxIS. The estimated amino acid preferences were consistent with previous knowledge about the IRF5 binding surface. Positions occupied by phosphorylatable serine residues exhibited a high frequency of aspartate and glutamate, likely because their negatively charged side chains are similar to phosphoserine.

<meta charset="utf8"></head><body>Forfatter Spotlight: En beregningsmæssig tilgang til at dechifrere aminosyrepræferencer i multispecifikke protein-protein-interaktioner

We describe a methodology based on sequence diversification to estimate the amino acid preferences of multispecific binding sites in protein-protein interactions (PPIs). In this strategy, thousands of potential peptide ligands are generated and screened in silico, thus overcoming some limitations of available experimental methods.