Name: the use of a &#946;-lactamase-based conductimetric biosensor assay to detect biomolecular interactions
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Description: Watch this Scientific Journal Video about The Use of a ÃŽÂ²-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions at JoVE.com

Forfatterne bekrefter Vallonsk Region i Belgia innenfor rammen av forskningsprosjekter SENSOTEM og NANOTIC samt National midler For the forskning (F.R.S.-F.N.R.S) for deres støtte.

1. protein eksempel forberedelse
<ol><li>Produsere og rense hybrid protein BlaP-cAb-Lys3 som rapportert i våre tidligere studie15. Lagre protein i 50 mM fosfat buffer pH 7.4 med følgende sammensetning: 8 g NaCl, 0.2 g KCl, 1,44 g Na2HPO4 og 0,24 g KH2PO4 oppløst i 800 mL destillert vann fikse pH av løsningen 7.4 før justere det siste bindet av løsningen 1 sterilisere L. Filter protein løsningen.</li>
	<li>Forberede en høne egg hvit lysozyme (HE...

<ol>
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H., Laurent, A. H., Hilser, V. J., Ostermeier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+protein+switches+based+on+an+ensemble+model+of+allostery.">Design of protein switches based on an ensemble model of allostery.</a> Nat Commun. 6, 6968 (2015).</li><li>Collinet, B., 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=Functionally+accepted+insertions+of+proteins+within+protein+domains.">Functionally accepted insertions of proteins within protein domains.</a> J Biol Chem. 275, 17428-17433 (2000).</li><li>Betton, J. M., Jacob, J. P., Hofnung, M., Broome-Smith, J. 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I dette arbeidet presenterer vi en metode for å functionalize en nanobody med BlaP β-lactamase som en operatør protein og vi vise at vi lykkes kan implementere resulterende hybrid protein i en potentiometric sensor analysen. Det viktigste nyvinning aspektet av vårt arbeid i forhold til andre biosensor analyser er kovalente koblingen av antistoff delen som den enzymatiske aktiviteten som genererer elektrisk signal. Denne såkalte protein innsetting teknologien presenterer fordeler og begrensninger som vil være hovedf...

Biosensors er analytiske enheter som kombinerer en bio-molekylær interaksjon med fysisk eller kjemiske signalnettverk enheter kalles transdusere1. Registrert signalene kan deretter tolkes og for å overvåke samspillet mellom partnerne immobilisert og gratis. De fleste av biosensors innebærer bruk av et antistoff å oppdage analytter som hormoner eller annen patogen markører2. Skjellige formater kan brukes og inkludere masse-baserte, magnetisk, optisk eller elektrokjemisk biosensors. Sistnevnte er blant de mest brukte sensorer og fungerer ved å konvertere en bindende hendelse til et elektrisk signal. De forestillinger og sensitiviteten for alle antistoff-baserte biosensors er sterkt avhengig av hovedsakelig to parametere: i) kvaliteten på antistoffer og ii) egenskapene for systemet brukes til å generere signal2.
Antistoffer er høymolekylær masse dimeric proteiner (150-160 kDa) som består av to tunge kjeder og to lys kjeder. Samspillet mellom lette og tunge kjedene er hovedsakelig stabilisert hydrofobe samhandling samt en bevart disulfide obligasjon. Hver kjede omfatter en variabel domene som samhandler med antigen i hovedsak via tre hypervariable regioner heter komplementære bestemme regioner (CDR1-2-3). Til tross for mange fremskritt i feltet store uttrykket av full lengde antistoffer med rimelig uttrykk systemer (f.eks E. coli) ofte fører til produksjon av ustabil og aggregert proteiner. Det er derfor ulike antistoff fragmenter har blitt utviklet som enkelt-kjeden variabel fragmenter3 (ScFvs ≈ 25 kDa). De består av variabel domener av henholdsvis en tunge og en lys kjeder som covalently er knyttet sammen av en syntetisk aminosyresekvens. Men disse fragmentene ofte viser en dårlig stabilitet og har tendens til å samle, siden de viser en stor del av sine hydrofobe regioner til løsemiddel4. I denne sammenheng synes enkelt kjede Kamelid antistoff fragmenter, kalles nanobodies eller VHHs, å være gode alternativer til ScFvs. Disse domenene korresponderer til variabel domener av Kamelid single-kjeden antistoffer. I motsetning til konvensjonelle antistoffer, Kamelid antistoffer er blottet for lys kjeder og bare inneholde to tunge kjeder5. Nanobodies er derfor minste monomerisk antistoff fragmenter (12 kDa) kunne binde til et antigen med tilhørighet lik som konvensjonelle antistoffer6. I tillegg presenterer de forbedret stabilitet og løselighet sammenlignet med andre fulle antistoffer eller antistoff fragmenter. Til slutt, deres små størrelser og deres utvidet CDR3 looper tillate dem å gjenkjenne kryptiske epitoper og binder seg til enzymet aktive nettsteder7,8. I dag er disse domenene får betydelig oppmerksomhet og har vært sammen til biosensor teknologi. For eksempel Huang et al. har utviklet en nanobody-baserte biosensor av kvantifisering av menneskelig prostata-spesifikt antigen (PSA)9.
Som nevnt over er en viktig parameter biosensor analyser effektiviteten til systemet brukes til å generere elektrisk signal. Derfor enzym-baserte elektrokjemisk biosensors har tiltrukket seg stadig økende oppmerksomhet og har vært brukt mye for ulike applikasjoner som helse, matsikkerhet og miljøovervåking. Disse biosensors er avhengige av katalytisk hydrolyse av et av et enzym å generere elektrisk signal. I denne sammenheng ble β-lactamases vist å være mer spesifikk, mer følsomme og lettere å implementere eksperimentelt enn mange andre enzymer som alkalisk fosfatase eller pepperrot peroxidase10. Β-lactamases er enzymer som er ansvarlig for bakteriell motstand mot β-Laktam antibiotika av hydrolyzing dem. De er monomerisk, veldig stabil, effektiv, og liten størrelse. Videre genererer domene/peptid innsettinger i β-lactamases bi-fungerende chimeric proteiner som ble vist å være effektive verktøy å studere protein-ligand interaksjoner. Faktisk har studier vist at innsetting av antistoff variabel TEM1 β-lactamase resultatene i et chimeric protein som fortsatt stand til å binde med høy affinitet til sine mål antigen. Interessant, ble antigen bindingen vist å indusere allosteric regulering av TEM1 katalytisk aktivitet11,12. Videre viste vi i flere studier at protein domene innsetting en ettergivende løkke av Bacillus licheniformis BlaP β-lactamase genererer funksjonelle chimeric proteiner som er godt egnet til å overvåke protein-ligand interaksjoner13 ,14. Vi nylig satt inn en nanobody, kalt cAb-Lys3, i denne ettergivende innsetting site BlaP15. Denne nanobody ble vist å binde til hen-egg-hvit lysozyme (HEWL) og til å hemme den enzymatiske aktiviteten16. Vi viste at generert hybrid protein, kalt BlaP-cAb-Lys3, beholdt en høy spesifisitet / affinitet mot HEWL mens β-lactamase aktiviteten forble uendret. Vi har kombinert β-lactamase hybridteknologi å en elektrokjemisk biosensor og viste at mengden av genererte elektrisk signal var avhengige av samspillet mellom BlaP-cAb-Lys3 og HEWL immobilisert på en elektrode. Faktisk induserer hydrolyse av β-Laktam antibiotika av BlaP en proton utgivelse som kan konverteres til en kvantitativ elektrisk signal. Denne kombinasjonen av β-lactamase hybridteknologi med en elektrokjemisk biosensor er rask, følsom, kvantitativ, og gir sanntids måling av genererte signalet. Denne metodikken er beskrevet her.

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			<td height="21" style="height:21px;width:515px;">Reagents</td>
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			<td>Sigma-Aldricht</td>
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		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">K2HPO4</td>
			<td>Sigma-Aldricht</td>
			<td>1551128</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Sigma-Aldricht</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris&#8211;HCl</td>
			<td>Roche</td>
			<td>10812846001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA&#160;</td>
			<td>Sigma-Aldricht</td>
			<td>E9884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>Sigma-Aldricht</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2HPO4&#160;</td>
			<td>Sigma-Aldricht</td>
			<td>NIST2186II</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-mercaptoethanol</td>
			<td>Sigma-Aldricht</td>
			<td>M6250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">alanine</td>
			<td>Sigma-Aldricht</td>
			<td>A7627</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">HClO4</td>
			<td>Fluka</td>
			<td>34288</td>
			<td>1M HClO4 solution, distributor : Sigma-Aldricht</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">casein hydrolysate</td>
			<td>Sigma-Aldricht</td>
			<td>22090</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">benzylpenicillin sodium</td>
			<td>Sigma-Aldricht</td>
			<td>B0900000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">hen egg white lysozyme</td>
			<td>Roche</td>
			<td>10837059001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">heptane</td>
			<td>Sigma-Aldricht</td>
			<td>246654</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">methanol</td>
			<td>Sigma-Aldricht</td>
			<td>322415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ammonium hydroxide solution</td>
			<td>Sigma-Aldricht</td>
			<td>380539</td>
			<td>28% NH3&#160;in H2O, purified by double-distillation (concentrated?)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laboratory consumables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-well plate&#160;</td>
			<td>Greiner Bio-One</td>
			<td>657165</td>
			<td>CELLSTAR 6-Well Plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pH meter</td>
			<td>WTW</td>
			<td>1AA110</td>
			<td>Lab pH meter inoLab pH 7110</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">vacuum and filtration system</td>
			<td>Nalgene</td>
			<td>NALG300-4100</td>
			<td>Filter holders with receiver, distributor : VWR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">potentiometric sensor chips</td>
			<td></td>
			<td></td>
			<td>manufactured by Yunus and colleagues (ref 16)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PGSTAT30 Autolab</td>
			<td>Metrohm Autolab</td>
			<td></td>
			<td>discontinued, succesor Autolab PGSTAT302N</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">digital multimeter, METRAHit 22M</td>
			<td>Gossen Metrawatt</td>
			<td></td>
			<td>discontinued, successor Metrahit Base</td>
		</tr>
	</tbody>
</table>

the use of a &#946;-lactamase-based conductimetric biosensor assay to detect biomolecular interactions

Biosensors blir stadig mer viktig og implementert i ulike felt som patogen gjenkjenning, molekylær diagnose, miljøovervåking og mat sikkerhetskontroll. I denne sammenhengen brukte vi β-lactamases som effektiv reporter enzymer i flere protein-protein samhandling studier. Deres evne til å akseptere innsettinger peptider eller strukturert proteiner/domener sterkt oppfordrer videre bruk av disse enzymene generere chimeric proteiner. I en fersk studie inn vi et enkelt domene antistoff fragment Bacillus licheniformis BlaP β-lactamase. Disse små domener, også kalt nanobodies, er definert som antigen bindende domener av enkelt kjede antistoffer fra camelids. Som vanlig dobbel kjeden antistoffer viser de høy slektskap og særegenheter for sine mål. Resulterende chimeric protein utstilt høy affinitet mot målet samtidig beholde β-lactamase aktiviteten. Dette tyder på at de nanobody og β-lactamase moieties fortsatt fungere. I den nåværende arbeidet rapportere vi en detaljert protokoll som kombinerer hybrid β-lactamase systemet biosensor teknologien. Bestemt binding av nanobody til målet kan oppdages takket være en conductimetric måling av protoner utgitt av katalytisk aktiviteten av enzymet.

Design og utvikling av chimeric protein BlaP-cAb-Lys3
Figur 1 representerer innsetting av cAb-Lys3 i en ettergivende løkke av BalP klasse A β-lactamase fra Bacillus licheniformis. Innsetting ble utført mellom rester Asp198 og Lys199. Et trombin cleavage område ble introdusert på hver side av cAb-Lys3. Celler forvandlet en grunnleggende uttrykk plasmider koding BlaP-cAb-Lys3 chimeric protein kunne vokse i nærvær av e...

Watch this Scientific Journal Video about The Use of a ÃŽÂ²-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions at JoVE.com

The Use of a ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

I dette arbeidet rapporterer vi en ny metode for å studere protein-protein interaksjoner ved hjelp av en conductimetric biosensor basert på hybridteknologi β-lactamase. Denne metoden er avhengig av utgivelsen av protoner ved hydrolyse av β-lactams.

Bruk av en β-lactamase-baserte Conductimetric Biosensor analysen å oppdage Biomolecular interaksjoner

Biosensors are becoming increasingly important and implemented in various fields such as pathogen detection, molecular diagnosis, environmental ...

Research

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Bioengineering

The Use of a &#946;-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions

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Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Fastsettelse av Transport Valuta xenobiotics og nanomaterialer over placenta ved hjelp<em&gt; Ex vivo</em&gt; Menneskelig placentaperfusjon Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Bruk av Merkfritt Optiske biosensorer til Detect Modulation av kaliumkanaler av G-protein koblede reseptorer

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

Bruk av en High-throughput<em&gt; In Vitro</em&gt; Mikrofluid System for å utvikle muntlige Multi-arter Biofilm

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

Use of a Rat Model to Study Ventral Abdominal Hernia Repair

Bruk av en rotte modell å studere ventrale Abdominal brokk reparasjon

Ventral abdominal hernia is a relatively common clinical condition that sometimes requires herniorraphy (surgical repair). The repair of ventral abdominal ...

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

PIP-on-a-chip: En etikettfri studie av protein-fosfinositid-interaksjoner

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

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In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

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Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...