Gli autori desiderano ringraziare il Dr. David Tirrell (Caltech) per la sua gentile dono del plasmide pQE9 AC 10 ATRP 12, il Dr. Tom Muir (Princeton University) per la sua gentile dono del plasmide kanr-IntRBS-NpuNC-CFN 11, Dr. Takehisa Matsuda (Kanazawa Institute of Technology, Hakusan, Ishikawa, Giappone) per la sua gentile dono del plasmide PET30-Cuta-Tip1 10, e il Dr. Jay D. Keasling (UC Berkley) per la sua gentile dono del plasmide pJD757 13 . Questo lavoro è stato sostenuto in parte dalla National Science Foundation CARRIERA, US Air Force YIP e Norman Hackman Programma di ricerca avanzata.

1. Plasmide Costruzioni NOTA: Tutti i geni sono stati amplificati in reazioni di PCR standard utilizzando Phusion-High Fidelity DNA Polymerase secondo le specifiche del produttore. Primer utilizzati per il clonaggio sono stati descritti in precedenza 9. Tutti i costrutti sono elencati nella Tabella 1. <ol><li> Per generare Cuta-NpuN (N, Tabella 1): <ol><li> PCR amplifica Cuta e NpuN geni da plasmidi PET30-Cuta-Tip1 10 e kanr-IntRBS-NpuNC-CFN 11, rispettivamente, utilizzando i primer appropriati. </li><li> Digerire questi frammenti con gli appropriati enzimi di restrizione e sequenzialmente inserire questi frammenti nel vettore pET26b tra il promotore T7 e un tag 6xHistidine C-terminale per generare N (Figura 2A). </li></ol></li><li> Per generare NpuC-S-Cuta (C, tabella 1): <ol><li> PCR amplificare NpuC, Cuta e S frammentazionet [AG 3 (PEG)] 10 dal plasmide kanr-IntRBS-NpuNC-CFN 11, PET30-Cuta-Tip1 10 e pQE9 AC 10 ATRP 12, rispettivamente, utilizzando i primer appropriati. </li><li> Digerire questi frammenti con gli appropriati enzimi di restrizione e sequenzialmente inserire questi frammenti nel vettore pET26b tra promotore T7 e un 6xHistidine C-terminale per generare C (Figura 2B). </li></ol></li><li> Per generare NpuC-S-SH3 lig-Cuta (C-SH3 lig, Tabella 1): <ol><li> PCR Amplificazione Cuta utilizzando primer contenenti un lig SH3 (PPPALPPKRRR) ed un linker flessibile (GGGGS) 2 per generare frammento SH3 lig-Cuta. </li><li> Sostituire il gene Cuta da C con frammento SH3 lig-Cuta. </li></ol></li><li> Per generare SH3-GFP (Tabella 1): <ol><li> Amplificare il gene SH3 dal plasmide pJD757 13 utilizzando ilprimer appropriati. </li><li> Fondere questo frammento di gene GFP e inserirlo nel vettore pET26b tra il promotore T7 (Figura 2C) e un tag 6xHistidine C-terminale. </li></ol></li></ol> 2. Protein Expression <ol><li> Trasformare 50 ml di chimicamente competenti di Escherichia coli BL21 (DE3) con il plasmide di espressione appropriato. </li><li> Dopo la trasformazione, serialmente diluire queste cellule, e piatto su Luria-Bertani (LB) / piastre di agar contenenti 50 ug / ml kanamicina. </li><li> Incubare le piastre contenenti cellule trasformate a 37 ° C per ~ 15 ore. </li><li> Dopo l&#39;incubazione, scegliere la piastra contenente 50-100 colonie e risospendere tutte le colonie in 5 ml di brodo LB. </li><li> Sospensione Trasferimento a 1 L di brodo LB contenente kanamicina (50 mcg / ml) e crescere le cellule a 37 ° C con agitazione a 250 rpm. Monitorare l&#39;assorbanza a 600 nm (OD 600). Far crescere la cultura fino OD 600 ~ 0.8. <ol><li> Per C e C-SH3 lig, indurre l&#39;espressione della proteina aggiungendo isopropil β-D-1-thiogalactopyranoside (IPTG) alla coltura (1 mM concentrazione finale) e incubare la coltura a 37 ° C per 4 ore sotto agitazione a 250 rpm. </li><li> Per N e SH3-GFP, raffreddare la cultura a ~ 18 ° C immergendo il pallone di coltura in un bagno di acqua ghiacciata per ~ 5 min. Indurre l&#39;espressione di proteine ​​con l&#39;aggiunta di IPTG alla cultura (concentrazione finale 1 mM) e incubare la coltura a 18 ° C per 14-18 ore agitando a 250 rpm. </li></ol></li><li> Dopo l&#39;espressione della proteina, centrifugare la cultura a 6.000 xg per 20 min a 4 ° C per raccogliere il pellet. Pellet cellulare conservare a -80 ° C fino all&#39;utilizzo. </li></ol> 3. Protein Purification <ol><li> Purificazione di N (condizioni denaturanti) <ol><li> Risospendere pellet cellulari in tampone A (Tabella 2) a 10 ml / g di pellet bagnato. </li><li> Immersposta la sospensione pellet in un bagno di ghiaccio-acqua e distruggere le cellule mediante sonicazione (Amp 10, con 1 sec impulso e 6 sec pausa di 1 min). </li><li> Centrifugare il lisato a 16.000 xg per 20 min a 4 ° C. </li><li> Scartare il surnatante. Risospendere il pellet in Buffer DA (contenente 8 M urea) e centrifugare la sospensione a 16.000 xg per 20 min a 4 ° C. </li><li> Passare il surnatante con una colonna di 5 ml di acido Ni-nitrilotriacetico (NTA) precedentemente equilibrata con tampone DA. </li><li> Lavare colonna con 30 ml di tampone DA integrato con imidazolo 45 mM. Eluire proteina purificata utilizzando 20 ml di tampone DA integrato con imidazolo 150 mM. </li><li> Ridurre la concentrazione di urea nel campione ad &lt;1 mM da uno tra i seguenti metodi indicati in 3.1.7.1 o 3.1.7.2: <ol><li> Dializzare proteina in tampone DPBS (Tabella 2) a 4 ° C per una notte usando tubi con &lt;20 kDa cutoff. </li><li> Centrifugare purificato proteina in un 30 &amp; #160; kDa ultra-filtrazione centrifuga colonna a 2.800 xg, a 4 ° C fino a quando il volume è inferiore a 1 ml. Aggiungere 14 ml tampone DPBS alla colonna di diluire il campione di proteine. Ripetere la centrifugazione / diluizione passaggi da altre tre volte. </li></ol></li><li> Dopo lo scambio di tamponi, aggiungere ditiotreitolo (DTT) per la proteina purificata (finale 2 mM) e concentrato proteico di ~ 100 mg / ml mediante centrifugazione attraverso un ultra-filtrazione centrifuga colonna 30 kDa a 2800 xg, a 4 ° C. </li><li> Aliquotare proteica concentrata e conservare a -80 ° C fino all&#39;utilizzo. </li></ol></li><li> Purificazione di C e C-SH3 lig (condizione nativo) <ol><li> Risospendere pellet cellulari in tampone B (pH 6,0) (Tabella 2) supplementato con 1x cocktail di inibitori delle proteasi a 10 ml / g di pellet bagnato. Utilizzare tampone acido a minimizzare la degradazione proteolitica della proteina bersaglio. </li><li> Perturbare sospensione cellulare mediante sonicazione come descritto in 3.1.2. Centrifugare del Lysmangiato a 16.000 xg per 20 min a 4 ° C e conservare il surnatante. </li><li> Passare il lisato solubile attraverso una colonna 5 ml Ni-NTA precedentemente equilibrata con tampone B. </li><li> Lavare colonna con tampone B supplementato con imidazolo 45 mM, ed eluire la proteina bersaglio in 20 ml di tampone B supplementato con imidazolo 150 mM. </li><li> Per la C, passare al punto 3.2.6. Per la C-SH3 lig, effettuare una purificazione a scambio ionico ulteriore passo per rimuovere proteine ​​parzialmente degradata come indicato nei punti 3.2.5.1 alla 3.2.5.3 <ol><li> Ridurre la concentrazione di NaCl in C-SH3 lig a &lt;1 mm seguendo la procedura descritta in 3.1.7. </li><li> Caricare la proteina bersaglio su un ml scambiatore anionico perline agarosio colonna 5 matrice precedentemente equilibrata con tampone sodio fosfato (50 mM, pH 7,0). </li><li> Eluire obiettivo della proteina dalla colonna eseguendo un gradiente da una soluzione contenente 10 mM Tris-HCl pH 8.0 buffer per una soluzione contenente lo stesso tampone supplementato con 1 M NaCl. Prelevare campioni durante proteine ​​eluizione e campioni piscina con la massima purezza basato su sodio dodecil solfato elettroforesi su gel di poliacrilammide (SDS-PAGE). </li></ol></li><li> Buffer scambiare la proteina purificata in tampone DPBS, come descritto in 3.1.7. </li><li> Aggiungere DTT alla proteina purificata (concentrazione finale 2 mM) e concentrare la proteina di ~ 100 mg / ml usando un ultra-filtrazione colonnine 30 kDa come descritto in 3.1.8. Aliquotare e conservare concentrate proteine ​​a -80 ° C fino all&#39;utilizzo. </li></ol></li><li> Purificazione di SH3-GFP <ol><li> Pellet cellulari Risospendere con tampone A a 10 ml / g di pellet bagnato. </li><li> Disrupt sospensione pellet mediante ultrasuoni come descritto in 3.1.2. </li><li> Centrifugare il lisato a 16.000 xg per 20 min a 4 ° C e raccogliere il surnatante. </li><li> Passare il surnatante (lisato solubile) attraverso una colonna 5 ml Ni-NTA precedentemente equilibrata conBuffer A. </li><li> Lavare colonna con 30 ml di tampone A completato con imidazolo 45 mM. Eluire proteina purificata utilizzando 20 ml di tampone A, completata con imidazolo 150 mM. </li><li> Buffer scambiare la proteina purificata nel buffer DPBS utilizzando un approccio simile a quello descritto in 3.1.7 e concentrare la proteina di ~ 150 mg / ml utilizzando un ultra-filtrazione colonnine 30 kDa come descritto in 3.1.8. </li><li> Aliquotare e deposito proteico purificato a -80 ° C fino all&#39;utilizzo. </li></ol></li><li> SDS-PAGE analisi di campioni purificati contenenti Cuta <ol><li> Diluire ogni proteina purificata in acqua bidistillata per ridurre la concentrazione di NaCl a ~ 1 mM. A questa concentrazione di NaCl, la maggior parte delle proteine ​​Cuta Trimer gestita come monomeri su gel SDS-PAGE. </li><li> Mescolare campioni con tampone campione SDS 2x (0,5 M Tris-HCl, pH 6,8, 20% glicerolo, 10% w / v SDS, 0.1% w / v bromo-fenolo blu, 2% β-mercaptoetanolo), incubato a 95 ° C per 5 min. </li><li> Caricare il samcipi su un gel SDS-PAGE 12%. Effettuare elettroforesi ad una tensione costante di 200 V per ~ 50 min. </li><li> Osservare proteine ​​in gel mediante colorazione con blu brillante Coomassie R250 seguendo i protocolli standard (Figura 1C). </li></ol></li></ol> 4. Idrogel Formazione NOTA: i idrogel campione eseguite in questo studio contengono 1,6 mm di ogni blocco di idrogel salvo diversa indicazione. Questa concentrazione proteica produce un idrogel morbida e stabile. ATTENZIONE: Sodio azide (NaN 3) è aggiunto l&#39;idrogel ad una concentrazione finale di 0,5% w / v per prevenire la contaminazione batterica. NaN 3 è altamente tossico e deve essere maneggiata con estrema cura, come indicato nella scheda di dati di sicurezza del materiale. <ol><li> Calcolare il volume per ciascuna delle proteine ​​concentrate necessari per ottenere una concentrazione finale di 1,6 mM in un idrogel campione 100 microlitri. Per esempio: Concentrazione di N:100 mg / ml Peso molecolare di N: 26.3 kDa (vedi Tabella 1) Concentrazione 100 microlitri desiderata:: volume desiderato 1,6 mm <img alt="" fo:content-width="4.5in" fo:src="/files/ftp_upload/51202/book.JPG" src="/files/ftp_upload/51202/book.JPG" width="600" /></li><li> Per effettuare una microlitri idrogel 100 (1,6 mM), mescolare C (x ml, volume calcolato secondo 4.1) con 5% NaN 3 (10 ml), DTT 100 mM (5 ml) e N (y microlitri, volume calcolato secondo 4.1) in una fiala di vetro da 2 ml. </li><li> Aggiungi il tampone di DPBS ((85 - x - y) microlitri) per flaconcino per ottenere un volume finale di 100 ml e mescolare manualmente tutti i componenti attraverso un movimento rotatorio con un puntale. Nota: La soluzione diventa molto viscoso con la miscelazione. </li><li> Centrifugare la miscela per 2 minuti a 8000 xg per rimuovere le bolle d&#39;aria. </li><li> Incubare la miscela a temperatura ambientetemperatura durante la notte per permettere la reazione inteina trans-splicing per raggiungere il completamento. Confermare la formazione di idrogel girando il tubo a testa in giù. Le proteine ​​non affluiranno se si forma un idrogel. </li><li> Stima della resa inteina trans-splicing controllando campioni (0,5 microlitri ciascuno) raccolti prima fase 4.2 e 4.5 dopo passo su un gel SDS-PAGE, come descritto in 3.4 (Figura 1C). </li></ol> 5. Immobilizzazione della GFP via Docking Protein (DP) e Docking Station Peptide (DSP) Interazione <ol><li> Per fare un 50 microlitri GFP-funzionalizzato idrogel (1,2 mM), combinare C-SH3 lig (x microlitri, calcolato secondo 4.1) e SH3-GFP (y microlitri, calcolato secondo 4.1) in rapporto molare 1:1 in 1.7 ml provetta e incubare la miscela a temperatura ambiente per 30 min. </li><li> Aggiungere 5% NaN 3 (5 ml), DTT 100 mm (2,5 ml), (42.5 - x - &lt;em&gt; y) DPBS microlitri allo stesso tubo. Aggiungere N (y microlitri, calcolato secondo 4.1) per ottenere un rapporto 1:1 molare di N e C-SH3 lig. Mescolare il campione utilizzando un puntale da un moto vorticoso. </li><li> Centrifugare la miscela a 8.000 xg per 2 minuti e incubare la miscela a temperatura ambiente per una notte al buio. Un idrogel incapsulando forme SH3-GFP durante l&#39;incubazione. </li></ol> 6. L&#39;uso di 1.6 Hydrogel mM come impalcatura immobilizzazione per reazione enzimatica in solvente organico <ol><li> Utilizzare l&#39;HRP come modello enzima. Preparare una soluzione stock di HRP (28 mg / ml o 0,63 mm) a DPBS. </li><li> Per effettuare una microlitri idrogel 30 (1.6 mM) entrapping HRP, unire C (x microlitri, calcolato secondo 4.1) con HRP (2 ml), 5% NaN 3 (3 ml) e DTT (1,5 ml di 100 mM) all&#39;interno di un 1,7 ml provetta da centrifuga. </li><li> Aggiungi N (y &lt;/ Em&gt; microlitri, calcolato secondo 4.1) e DPBS (23.5 - x - y) microlitri. Mescolare con un puntale con un movimento vorticoso. </li><li> Centrifugare la miscela a 8.000 xg per 2 minuti e incubare a temperatura ambiente per una notte. ATTENZIONE: i reggenti utilizzati per il seguente saggio di attività sono altamente tossici. Utilizzare raccomandazioni di sicurezza specifiche per le corrispondenti Schede Dati di Sicurezza. </li><li> Per reazione enzimatica, immergere l&#39;idrogel in 1 ml di cocktail di reazione contenenti N, diammina N-dimetil-p-fenilene (5,8 mM), fenolo (5,8 mM) e tert-butil idroperossido (2,9 mM) in n-eptano 14. Interrompere manualmente il gel utilizzando un puntale per aumentare la superficie di contatto di idrogel e il solvente. </li><li> Controllo prodotto HRP, un indofenolo tipo dye, misurando l&#39;assorbanza ottica di campioni prelevati in tempi diversi a 546 nm in un lettore di piastre (Figura 5). </li> &lt;/ Ol&gt;

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In questo lavoro, abbiamo dimostrato la sintesi di un altamente stabile mediata da inteina idrogel proteina. L&#39;uso di una scissione consente inteina l&#39;idrogel essere condizionale formata in risposta alla miscelazione dei due componenti in fase liquida. In particolare, la scissione collega inteina covalentemente due blocchi in fase liquida tramite una reazione di trans-splicing, producendo una unità polipeptide fiancheggiato da reticolazione unità che a sua volta sé ​​assembla in un idrogel. La fo...

Gli idrogel interamente in proteine ​​portano il potenziale per far avanzare in modo significativo campi diversi come l&#39;ingegneria dei tessuti, somministrazione di farmaci e biofabrication 1. Essi offrono vantaggi rispetto ai tradizionali idrogel polimerici sintetici, tra cui biocompatibilità e la possibilità di sostenere in modo non invasivo l&#39;incorporazione di proteine ​​globulari bioattive. In questo lavoro, si descrive lo sviluppo di una proteina idrogel romanzo formata tramite split-mediata da inteina reazione proteina trans-splicing e la sua applicazione come impalcatura immobilizzazione di proteine ​​(Figura 1). Gli elementi fondamentali per questo idrogel sono due blocchi copolimeri proteine ​​comprendenti ciascuno il frammento N-o C-terminale di una inteina suddivisa (IN e IC) e una subunità di una proteina multimerica reticolante. Il DnaE inteina da Nostoc punctiforme (Npu) è stato utilizzato come inteina suddivisa 2,3 e una piccola proteina trimeric (12 kDa) Cuta da Pyrococcus horikoshii &lt;/ Em&gt; è stato utilizzato come 4,5 proteina reticolante. Diversi reticolanti sono uniti attraverso inteina reazione catalizzata trans-splicing, che porta alla formazione di una rete di proteine ​​altamente reticolato (idrogel). Npu inteina è stato scelto per le sue cinetica di reazione rapida (t 1/2 = 63 sec) e ad alto rendimento trans-splicing (vicino al 80%) 2,3. La proteina Cuta stato scelto come reticolante grazie alla sua elevata stabilità. Trimeri Cuta hanno una temperatura di denaturazione di prossimità 150 ° C e mantenere la struttura quaternaria trimeric in soluzioni contenenti fino al 5 M guanidina cloridrato 4,6. Dal momento che lo scambio tra le diverse subunità crosslinkers è un importante contributo della idrogel fisica superficie di erosione 7, molto forte interazione tra subunità in Cuta dovrebbe scoraggiare tali scambi subunità, portando ad un idrogel più stabile. Uno di questi blocchi contiene anche un peptide altamente idrofilo S-frammento come mid-block per facilitare acquaritenzione 8. Miscelazione dei due blocchi idrogel inizio a una reazione trans-splicing tra IN e IC inteina frammenti, generando una catena polipeptidica più con reticolanti a entrambi i terminali. Reticolanti da più tali unità molecolari interagiscono tra loro, formando una rete idrogel altamente reticolato (Figura 1A). Una specifica &quot;docking station peptide&quot; (DSP) è incorporato in uno dei blocchi di idrogel per facilitare immobilizzazione stabile di un &quot;aggancio proteina&quot; (DP)-tag proteina bersaglio in idrogel. L&#39;uso di una inteina suddivisa per mediare il montaggio idrogel non solo fornisce una maggiore flessibilità per la sintesi proteica idrogel, ma permette anche ad alta densità, il caricamento uniforme della proteina bersaglio nell&#39;intero idrogel, come le proteine ​​target vengono caricati prima della formazione idrogel. La proteina idrogel mediata da inteina è altamente stazioneBLE in soluzione acquosa con poco a nessuna erosione rilevabile dopo 3 mesi a temperatura ambiente. La stabilità è mantenuto in una vasta gamma di pH (6-10) e temperature (4-50 ° C), e l&#39;idrogel è anche compatibile con solventi organici. Questo idrogel viene utilizzato per l&#39;immobilizzazione di due proteine ​​globulari: la proteina fluorescente verde (GFP) e la perossidasi di rafano (HRP). Idrogel intrappolare quest&#39;ultima proteina viene utilizzato per eseguire biocatalysis in un solvente organico.

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			Phusion High Fidelity DNA polymerase
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			New England BioLabs
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			M0530S
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			New England BioLabs
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			kanamycin sulfate
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			VWR
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			VWR
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			EM-5720
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			EM-9510
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			Dithiothreitol (DTT)
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			Fisher
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			BP172-5
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			Protease Inhibitor cocktail
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			Roche Applied Science
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			82020-066
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			Acros Organics
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			A0297990
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			Fisher
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			AC190380050
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			Caution, highly toxic
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			Horseradish peroxidase
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			Sigma
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			P8125-5KU
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			AC408460250
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			Caution, highly toxic
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			tert-butyl hydroperoxide
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			Caution, highly toxic
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			Sorvall
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			RC 6
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			Amicon Ultra
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			GE Healthcare Life Sciences
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synthesis of an intein-mediated artificial protein hydrogel

Presentiamo la sintesi di una proteina idrogel altamente stabile mediata da uno split-inteina catalizzata reazione proteina trans-splicing. Le componenti di questo idrogel sono due blocchi copolimeri proteine ​​contenenti ciascuno una subunità di una proteina trimeric che funge da reticolante e una metà di una inteina scissione. Una bobina casuale altamente idrofila viene inserito in uno dei blocchi copolimeri per la ritenzione idrica. Miscelazione dei due copolimeri a blocchi proteina innesca una reazione trans-splicing inteina, ottenendo una unità polipeptide con reticolanti alle due estremità che rapidamente auto-assembla in un idrogel. Questo idrogel è molto stabile sia in condizioni acide o basiche, a temperature fino a 50 ° C, e in solventi organici. L&#39;idrogel rapidamente riforme dopo la rottura shear-indotta. Costituzione di una &quot;docking station peptide&quot; in blocco dell&#39;edificio idrogel permette conveniente incorporazione di &quot;aggancio proteina&quot;-tagged proteine ​​bersaglio.L&#39;idrogel è compatibile con mezzi di crescita di coltura tissutale, supporta la diffusione di molecole 20 kDa, e consente l&#39;immobilizzazione di proteine ​​globulari bioattivi. L&#39;applicazione del idrogel proteine ​​mediata da inteina come biocatalizzatore compatibile organico-solvente è stato dimostrato incapsulando l&#39;enzima perossidasi di rafano e corroborare la sua attività.

Uno schema per mediata da inteina formazione proteica idrogel è presentato nella Figura 1A. Gli elementi costitutivi del idrogel sono la proteina copolimeri Cuta-NpuN (N) e NpuC-S-Cuta (C) (Figura 1A, Tabella 1). NpuN / C sono i N-/C-fragments del DnaE naturalmente inteina suddivisa da Nostoc punctiforme (Npu). Cuta è una proteina trimeric stabile da Pyrococcus horikoshii 4,5. Miscelazione di purificato N e C in presenza dell&#39;agente riducente DTT induce la form...

Watch this Scientific Journal Video about Synthesis of an Intein-mediated Artificial Protein Hydrogel at JoVE.com

Synthesis of an Intein-mediated Artificial Protein Hydrogel

Presentiamo la sintesi di un idrogel split-mediata da inteina proteina. Le componenti di questo idrogel sono due copolimeri proteine ​​contenenti ciascuno una subunità di una proteina trimeric che funge da reticolante e una metà di una inteina scissione. Miscelazione dei due copolimeri proteina innesca una reazione trans-splicing inteina, ottenendo una unità polipeptide che autoassembla in un idrogel. Questo idrogel è altamente pH e temperatura stabile, compatibile con solventi organici, e facilmente incorpora proteine ​​globulari funzionali.

Sintesi di una proteina artificiale mediata da inteina Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

synthesis-of-an-intein-mediated-artificial-protein-hydrogel

Research

JoVE Journal

Bioengineering

12.0K Views.  Texas A&M University, College Station.  Presentiamo la sintesi di un idrogel split-mediata da inteina proteina. Le componenti di questo idrogel sono due copolimeri proteine ​​contenenti ciascuno una subunità di una proteina trimeric che funge da reticolante e una metà di una inteina scissione. Miscelazione dei due copolimeri proteina innesca una reazione trans-splicing inteina, ottenendo una unità polipeptide che autoassembla in un idrogel. Questo idrogel è altamente pH e temperatura stabile, ...

Video: Synthesis of an Intein-mediated Artificial Protein Hydrogel

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

Sintesi, assemblaggio e caratterizzazione di film monostrato Protetta delle nanoparticelle d&#39;oro per Elettrochimica proteine ​​monostrato

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

Ricerca

Bioingegneria

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with conventional one-sample-at-a-time synthesis techniques, a more recent high-throughput approach has been developed enabling the synthesis and testing of large libraries of polyanhydrides1. This will facilitate more efficient optimization and design process of these biomaterials for drug and vaccine delivery applications. The method in this work describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. In this robotically operated method (Figure 1), linear actuators and syringe pumps are controlled by LabVIEW, which enables a hands-free automated protocol, eliminating user error. Furthermore, this method enables the rapid fabrication of micro-scale polymer libraries, reducing the batch size while resulting in the creation of multivariant polymer systems. This combinatorial approach to polymer synthesis facilitates the synthesis of up to 15 different polymers in an equivalent amount of time it would take to synthesize one polymer conventionally. In addition, the combinatorial polymer library can be fabricated into blank or protein-loaded geometries including films or nanoparticles upon dissolution of the polymer library in a solvent and precipitation into a non-solvent (for nanoparticles) or by vacuum drying (for films). Upon loading a fluorochrome-conjugated protein into the polymer libraries, protein release kinetics can be assessed at high-throughput using a fluorescence-based detection method (Figures 2 and 3) as described previously1. This combinatorial platform has been validated with conventional methods2 and the polyanhydride film and nanoparticle libraries have been characterized with 1H NMR and FTIR. The libraries have been screened for protein release kinetics, stability and antigenicity; in vitro cellular toxicity, cytokine production, surface marker expression, adhesion, proliferation and differentiation; and in vivo biodistribution and mucoadhesion1-11. The combinatorial method developed herein enables high-throughput polymer synthesis and fabrication of protein-loaded nanoparticle and film libraries, which can, in turn, be screened in vitro and in vivo for optimization of biomaterial performance.

This method describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries.

Sintesi combinatoria di e High-throughput di rilascio di proteine ​​da film polimerico e Biblioteche nanoparticelle

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with ...

An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the limited access to donor hearts. An example of a treatment to recover the function of the heart consists of the local delivery of drugs and bioactives from a hydrogel. In this paper a method is introduced to formulate and inject a drug-loaded hydrogel non-invasively and side-specific into the pig heart using a long, flexible catheter. The use of 3-D electromechanical mapping and injection via a catheter allows side-specific treatment of the myocardium. To provide a hydrogel compatible with this catheter, a supramolecular hydrogel is used because of the convenient switching from a gel to a solution state using environmental triggers. At basic pH this ureido-pyrimidinone modified poly(ethylene glycol) acts as a Newtonian fluid which can be easily injected, but at physiological pH the solution rapidly switches into a gel. These mild switching conditions allow for the incorporation of bioactive drugs and bioactive species, such as growth factors and exosomes as we present here in both in vitro and in vivo experiments. The in vitro experiments give an on forehand indication of the gel stability and drug release, which allows for tuning of the gel and release properties before the subsequent application in vivo. This combination allows for the optimal tuning of the gel to the used bioactive compounds and species, and the injection system.

Supramolecular hydrogelators based on ureido-pyrimidinones allow full control over the macroscopic gel properties and the sol&#8211;gel switching behavior using pH. Here, we present a protocol for formulating and injecting such a supramolecular hydrogelator via a catheter delivery system for local delivery directly in relevant areas in the pig heart.

Un supramolecolare idrogel iniettabile and Drug-caricato per catetere iniezione locale nel cuore di maiale

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the ...

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel capabilities to perform basic and applied sciences in test tube reactions. Over the past decade, cell-free TXTL has become a powerful technique for a broad range of novel multidisciplinary research areas related to quantitative and synthetic biology. The new TXTL platforms are particularly useful to construct and interrogate biochemical systems through the execution of synthetic or natural gene circuits. In vitro TXTL has proven convenient to rapidly prototype regulatory elements and biological networks as well as to recapitulate molecular self-assembly mechanisms found in living systems. In this article, we describe how infectious bacteriophages, such as MS2 (RNA), &#934;&#935;174 (ssDNA), and T7 (dsDNA), are entirely synthesized from their genome in one-pot reactions using an all Escherichia coli, cell-free TXTL system. Synthesis of the three coliphages is quantified using the plaque assay. We show how the yield of synthesized phage depends on the biochemical settings of the reactions. Molecular crowding, emulated through a controlled concentration of PEG 8000, affects the amount of synthesized phages by orders of magnitudes. We also describe how to amplify the phages and how to purify their genomes. The set of protocols and results presented in this work should be of interest to multidisciplinary researchers involved in cell-free synthetic biology and bioengineering.

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System

A new generation of cell-free transcription-translation platforms has been engineered to construct biochemical systems in vitro through the execution of gene circuits. In this article, we describe how bacteriophages, such as MS2, &#934;&#935;174, and T7, are synthesized from their genome using an all E. coli cell-free TXTL system.

Sintesi dei batteriofagi infettive in un e. coli -basato sistema di espressione senza cellula

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel ...

An Additive Manufacturing Technique for the Facile and Rapid Fabrication of Hydrogel-based Micromachines with Magnetically Responsive Components

Polyethylene glycol (PEG)-based hydrogels are biocompatible hydrogels that have been approved for use in humans by the FDA. Typical PEG-based hydrogels have simple monolithic architectures and often function as scaffolding materials for tissue engineering applications. More sophisticated structures typically take a long time to fabricate and do not contain moving components. This protocol describes a photolithography method that allows for facile and rapid microfabrication of PEG structures and devices. This strategy involves an in-house developed fabrication stage that allows for the rapid fabrication of 3D structures by building upwards in a layer-by-layer fashion. Independent moving components can also be aligned and assembled onto support structures to form integrated devices. These independent components are doped with superparamagnetic iron oxide nanoparticles that are sensitive to magnetic actuation. In this manner, the fabricated devices can be actuated using external magnets to yield movement of the components within. Hence, this technique allows for the fabrication of sophisticated MEMS-like devices (micromachines) that are composed entirely out of a biocompatible hydrogel, able to function without an onboard power source, and respond to a contact-less method of actuation. This manuscript describes the fabrication of both the fabrication set-up as well as the step-by-step method for the microfabrication of these hydrogels-based MEMS-like devices.

An additive manufacturing strategy for processing UV-crosslinkable hydrogels has been developed. This strategy allows for the layer-by-layer assembly of microfabricated hydrogel structures as well as the assembly of independent components, yielding integrated devices containing moving components that are responsive to magnetic actuation.

Un additivo tecnica di fabbricazione per la Facile e rapida realizzazione di Micromachines base di idrogel con componenti magneticamente reattivi

Polyethylene glycol (PEG)-based hydrogels are biocompatible hydrogels that have been approved for use in humans by the FDA. Typical PEG-based hydrogels ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

Fabbricazione di una matrice di multiplex artificiale microambiente cellulare

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with controlled pore structure and mechanical properties. Cellulose nanocrystals (CNCs, 69.62 wt%) based hydrogel ink with matrix (sodium alginate and gelatin) was developed and 3D printed into scaffolds with uniform and gradient pore structure (110-1,100 &#181;m). The scaffolds showed compression modulus in the range of 0.20-0.45 MPa when tested in simulated in vivo conditions (in distilled water at 37 &#176;C). The pore sizes and the compression modulus of the 3D scaffolds matched with the requirements needed for cartilage regeneration applications. This work demonstrates that the consistency of the ink can be controlled by the concentration of the precursors and porosity can be controlled by the 3D printing process and both of these factors in return defines the mechanical properties of the 3D printed porous hydrogel scaffold. This process method can therefore be used to fabricate structurally and compositionally customized scaffolds according to the specific needs of patients.&#160;

The three critical steps of this protocol are i) developing the right composition and consistency of the cellulose hydrogel ink, ii) 3D printing of scaffolds into various pore structures with good shape fidelity and dimensions and iii) demonstration of the mechanical properties in simulated body conditions for cartilage regeneration.

3D stampato cellulosa porosa dell'idrogelo Nanocomposite ponteggi

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with ...

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an attractive alternative to liposomes or fatty acid-based vesicles. Externally or within the vesicles, peptides can be easily expressed and simplify the synthesis of membrane precursors. Provided here is a protocol for the creation of vesicles with diameters of ~200 nm based on the amphiphilic elastin-like polypeptides (ELP) utilizing dehydration-rehydration from glass beads. Also presented are protocols for bacterial ELP expression and purification via inverse temperature cycling, as well as their covalent functionalization with fluorescent dyes. Furthermore, this report describes a protocol to enable the transcription of RNA aptamer dBroccoli inside ELP vesicles as a less complex example for a biochemical reaction. Finally, a protocol is provided, which allows in vesiculo expression of fluorescent proteins and the membrane peptide, whereas synthesis of the latter results in vesicle growth.

Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.

In Vesiculo Synthesis of Peptide Membrane Precursori per la crescita autonoma della vescicola

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...

An Open Source Technology Platform to Manufacture Hydrogel-Based 3D Culture Models in an Automated and Standardized Fashion

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These issues represent drawbacks in workflows that can ultimately result in irreproducibility of research findings. Manual-based workflows are further limiting the advancement and widespread adoption of viscous materials, such as hydrogels used for biomedical applications. These challenges can be overcome by using automated workflows with standardized mixing processes to increase reproducibility. In this study, we present step-by-step instructions to use an open source protocol designer, to operate an open source workstation, and to identify reproducible mixtures. Specifically, the open source protocol designer guides the user through the experimental parameter selection and generates a ready-to-use protocol code to operate the workstation. This workstation is optimized for pipetting of viscous materials to enable automated and highly reliable handling by the integration of temperature docks for thermoresponsive materials, positive displacement pipettes for viscous materials, and an optional tip touch dock to remove excess material from the pipette tip. The validation and verification of mixtures are performed by a fast and inexpensive absorbance measurement of Orange G. This protocol presents results to obtain 80% (v/v) glycerol mixtures, a dilution series for gelatin methacryloyl (GelMA), and double network hydrogels of 5% (w/v) GelMA and 2% (w/v) alginate. A troubleshooting guide is included to support users with protocol adoption. The described workflow can be broadly applied to a number of viscous materials to generate user-defined concentrations in an automated fashion.

This protocol serves as a comprehensive tutorial for standardized and reproducible mixing of viscous materials with a novel open source automation technology. Detailed instructions are provided on the operation of a newly developed open source workstation, the usage of an open source protocol designer, and the validation and verification to identify reproducible mixtures.

Una piattaforma tecnologica open source per la produzione di modelli di cultura 3D basati su idrogel in modo automatizzato e standardizzato

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These ...

From Cells to Cell-Free Protein Synthesis within 24 Hours Using Cell-Free Autoinduction Workflow

Cell-free protein synthesis (CFPS) has grown as a biotechnology platform that captures transcription and translation machinery in vitro. Numerous developments have made the CFPS platform more accessible to new users and have expanded the range of applications. For lysate based CFPS systems, cell extracts can be generated from a variety of organisms, harnessing the unique biochemistry of that host to augment protein synthesis. Within the last 20 years, Escherichia coli (E. coli) has become one of the most widely used organisms for supporting CFPS due to its affordability and versatility. Despite numerous key advances, the workflow for E. coli cell extract preparation has remained a key bottleneck for new users to implement CFPS for their applications. The extract preparation workflow is time-intensive and requires technical expertise to achieve reproducible results. To overcome these barriers, we previously reported the development of a 24 hour cell-free autoinduction (CFAI) workflow that reduces user input and technical expertise required. The CFAI workflow minimizes the labor and technical skill required to generate cell extracts while also increasing the total quantities of cell extracts obtained. Here we describe that workflow in a step-by-step manner to improve access and support the broad implementation of E. coli based CFPS.

This work describes the preparation of cell extract from Escherichia coli (E. coli) followed by cell-free protein synthesis (CFPS) reactions in under 24 hours. Explanation of the cell-free autoinduction (CFAI) protocol details improvements made to reduce researcher oversight and increase quantities of cell extract obtained.