Gli autori desiderano ringraziare il Sig. Luis F. Delgadillo (Dipartimento di Ingegneria Chimica e Biomolecolare, Ohio University), il Sig. Matthew H. Williams (Dipartimento di Ingegneria Chimica e Biomolecolare, Ohio University) e il Dr. Filiberto Cedeno-Laurent (Brigham and Women's Hospital e Harvard Medical School) per l'assistenza tecnica di esperti. Gli autori desiderano inoltre ringraziare gli studenti dell'inverno 2011 CHE 404/BME 504 e dell'autunno 2012 CHE 4830/BME 5830 (Dipartimento di Ingegneria Chimica e Biomolecolare e Ingegneria Biomedica, Ohio University) per la consulenza tecnica e la discussione. Questo lavoro è stato supportato dalla sovvenzione per la strumentazione di ricerca principale della National Science Foundation (NSF) CBET-1039869 (MMB), NSF CBET-1106118 (MMB), Dermatology Foundation Research Grant A050422 (SRB), National Institutes of Health (NIH) NSC grant 1R15CA161 830-01 (MMB), NIH Kirschstein-NRSA Postdoctoral Fellowship F32CA144219-01A1 (SRB), NIH/NCI R01CA173610 (CJD) e NIH NCCAM grant R01AT004628 (CJD).

1. Verificare l'affinità per le proteine A
<ol><li>Verificare che la proteina che esprime fc (proteina di fusione ricombinante o anticorpo IgG, finora denominata "proteina") abbia un'affinità accettabile per la proteina A14-17 prima di utilizzare l'adsorbente della membrana A della proteina E e verificare la presenza della proteina nella soluzione stock(ad esempio, supernatante di coltura cellulare chiarificato dalla centrifugazione a 1.000 x g per 5 minuti a cellule sospese a pellet). Utilizzare citometria a flusso, western blotting, ELISA, ecc. per rilevare la funzione proteica e/o la presenza di Fc, in base alle preferenze di laboratorio. Idealmente, quantificare la proteina nel supernatante di coltura cellulare (fase 6) per evitare di superare la capacità di adsorbente della membrana A della proteina. Procedere se viene rilevata una proteina.</li>
</ol>2. Preparare proteine A membrana adsorbente e coltura cellulare supernatante
<ol><li>Seguire le istruzioni del produttore per preparare l'adsorbente a membrana. Non lasciare mai che l'aria entri nell'adsorbente della membrana. Tutte le soluzioni perfuse attraverso l'adsorbente a membrana devono essere a temperatura ambiente e prefiltrate utilizzando un filtro da 0,22 μm. Riempire una siringa da 10 ml con la salina tamponata al fosfato (DPBS) filtrata da Dulbecco filtrata da 0,22 μm o tampone di scelta e scarico delle bolle d'aria. Perfondere DPBS per rimuovere la soluzione di archiviazione e equilibrare l'adsorbente a membrana immediatamente prima dell'uso.</li>
	<li>Filtrare il supernatante di coltura cellulare immediatamente prima della perfusione attraverso l'adsorbente a membrana, utilizzando un'unità di filtrazione sterile aspirata da 0,22 μm.</li>
</ol>3. Caricare la proteina A Membrane Adsorber
<ol><li>Aspirare il supernatante di coltura cellulare in una siringa Luer Lock (30 ml o più) e scaricare eventuali bolle d'aria.</li>
	<li>Assemblate materiali e attrezzature, come illustrato nella figura 1. Collegare la siringa all'ingresso dell'adsorbente della membrana. Collegare tubi flessibili all'uscita dell'adsorbente a membrana. Se lo si desidera, posizionare un filtro siringa da 0,22 μm tra la siringa e l'adsorbente a membrana. Utilizzare un pallone o una bottiglia per catturare il flusso attraverso (noto anche come filtrato), dall'adsorbente.</li>
	<li>Impostare la pompa della siringa sulla portata volumetrica desiderata, ma non superare la portata raccomandata dalproduttore (ad esempio 10 ml/min). Perfondere il supernatante della coltura cellulare attraverso l'adsorbente a membrana, raccogliendo il flusso attraverso un becher o un altro contenitore. Ricaricare la siringa con i supernatanti necessari.</li>
	<li>Se lo si desidera, testare il flusso attraverso per la presenza di proteine utilizzando citometria di flusso, western blotting, ELISA, ecc. in base alle preferenze di laboratorio. In genere non è necessario riperperperare il flusso attraverso.</li>
</ol>4. Proteina Elute dall'adsorbente di membrana
<ol><li>Lavare l'adsorbente a membrana con 10 ml di DPBS per rimuovere eventuali proteine non abbollate.</li>
	<li>Elute proteina dall'adsorbente di membrana alla portata desiderata(ad esempio 1 ml/min) utilizzando 10-15 ml di tampone di eluizione(ad esempio tampone di eluizione a base di ammina (pH 2,8)). Catturare l'eluato in un tubo contenentetampone neutralizzante (ad esempio 1 M Tris (pH 9,4)) al 10% del volume di eluizione (1,0-1,5 ml).</li>
	<li>In alternativa, la proteina eluta in un ml si trasforma in tubi contenenti 100 μl di tampone di neutralizzazione, utilizzando un volume totale di tampone di eluizione da 10-15 ml. Utilizzare il metodo di caratterizzazione preferito per testare ogni frazione per la presenza di proteine.</li>
	<li>Rigenerare l'adsorbente a membrana secondo le istruzioni del produttore. Perfondere 10 ml di DPBS filtrato da 0,22 μm o tampone di scelta, quindi 10 ml di DPBS filtrato da 0,22 μm in 50 mM naoh in 1 N NaCl e infine 10 ml di DPBS filtrato da 0,22 μm. Riempire l'adsorbente a membrana con il 20% di etanolo in DPBS per la conservazione a lungo termine a 4 ºC.</li>
</ol>5. Concentrare e dializzare la proteina
<ol><li>Depositare tutti gli eluati (o frazioni di eluizione contenenti proteine come determinato nella fase 4.3) in un'unità filtrante centrifuga cut-off a peso molecolare da 10 kDa. Seguire le istruzioni del produttore per la centrifugazione.</li>
	<li>Dializzare il ritensionato in un'unità di dialisi a piccolo volume (taglio del peso molecolare di 10 kDa) contro il tampone preferito, seguendo le istruzioni del produttore. Potrebbe essere necessario aggiungere un tampone al materiale dializzato se la precipitazione proteica è un problema. Se lo si desidera, il materiale dializzato può essere refrigerato fino a quando non è possibile eseguire il passaggio 6, ma non è consigliabile conservare a lungo termine senza conservanti.</li>
</ol>6. Quantificare e caratterizzare il prodotto purificato in una procedura di blotting occidentale accelerata
<ol><li>Risolvere gli standard di proteina purificata e Fc sul gel di scelta da parte di SDS-PAGE, in condizioni di riduzione o non riduzione comedesiderato 18-20. Utilizzare un mini gel anziché un gel midi per ridurre al minimo i tempi di esecuzione.<ol>
<li>Se non è già noto, determinare l'intervallo di standard Fc su quale segnale a banda varia linearmente rispetto alla quantità di carico(ad esempio 0,1-1 mg). Usa lo stesso gel, le condizioni di esecuzione, le condizioni di trasferimento e i reagenti che verranno utilizzati per quantificare e caratterizzare le proteine purificate.</li>
			<li>Caricare più quantità di campioni di proteine purificate, inclusi campioni diluiti con DPBS, per garantire che i segnali di banda proteica purificata si trovano all'interno dell'intervallo di segnale lineare degli standard Fc nell'analisi delle immagini.</li>
		</ol>
</li>
	<li>Trasferire le proteine dal gel a una membrana di fluoruro di polivinilidene (PVDF) in un sistema tampone discontinuo utilizzando un blotter semidry13, seguendo le istruzioni del produttore di blotter. Il tempo di trasferimento è in genere di 5-10 minuti. Se lo si desidera, Coomassie macchia il gel dopo il trasferimento per verificare l'efficienza deltrasferimento 21.</li>
	<li>Eseguire l'immunoblotting con anti-Fc o anti-IgG coniugato alla fosfatasi alcalina (AP) o alla perossidasi del ravanello del cavallo (HRP), in base alle preferenze di laboratorio, utilizzando un sistema di rilevamento delle proteine assistito dal vuoto. Il tempo di immunoblotting è in genere inferiore a 1 ora.</li>
	<li>Sviluppare immunoblot utilizzando il substrato e il metodo di scelta(ad esempio substrato AP e chemiluminescenza migliorata).</li>
	<li>Quantificare le proteine purificate mediante analisi delle immagini. Eseguire una regressione lineare sui segnali di banda dagli standard Fc. Se R2>0,90, utilizzare l'equazione per la linea per calcolare la quantità proteica dai suoi segnali di banda. Tenere conto della diluizione del campione, se necessario. Poiché la quantificazione della proteina viene eseguita sulla base di Fc, regolare il valore delle differenze di peso molecolare tra la proteina e Fc. Se R2<0,90, ripetere il passaggio 6 nella sua interezza.</li>
	<li>Convalidare proteine utilizzando saggi o metodi funzionali per rilevare Fc tramite citometria a flusso, western blotting, ELISA, ecc. in base alle preferenze di laboratorio.</li>
	<li>Diluire le proteine alla concentrazione desiderata e/o aggiungere eventuali conservanti o stabilizzanti desiderati alla soluzione proteica purificata prima di aliquota per la conservazione a lungo termine, congelata o meno.</li>
</ol>

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Non c'è nulla da rivelare.

Il protocollo qui descritto è stato sviluppato per isolare e caratterizzare rapidamente una proteina di fusione chimerica che esprime fc umano (Gal-1hFc), senza compromettere significativamente il recupero del prodotto o il costo di gonfiaggio. I componenti chiave del flusso di lavoro semplificato sono un adsorbente a membrana A proteico per l'isolamento e il trasferimento semidry e l'immunoblotting sottovuoto per la caratterizzazione da parte dell'assorbente occidentale.
La drastica diminuz...

L'isolamento di anticorpi e proteine ricombinanti che esprimono frammenti di immunoglobulina G (IgG) Fc dai supernatanti di coltura cellulare e dalle soluzioni cellulari diluite può essere realizzato utilizzando la cromatografia di affinità proteinA A. Nei laboratori e nell'industria scientifica e ingegneristica di base, le colonne piene di proteina porosa A agarosio, vetro o perline polimeriche sono più comunemente utilizzate per la cromatografia di affinità, nonostante gli elevati costi finanziari e i lunghi tempi dilavorazione 1-3. È molto apprezzato che la cromatografia di affinità rappresenti il più grande collo di bottiglia di spesa e lavorazione sia in laboratorio che in ambienteindustriale 2,4. Di conseguenza, sono stati sviluppati numerosi miglioramenti per ridurre i costi e i tempi di elaborazione senza sacrificare il recupero complessivo delprodotto dal treno di processo 1,2,5-7. Particolarmente promettente per l'isolamento di anticorpi e altre proteine che esprimono Fc è la cromatografia di affinità utilizzando una proteina A membrane adsorber2,5,7-10. Mentre la cattura fc nella cromatografia a colonna è limitata dalladiffusione (cioè la proteina che esprime fc deve diffondersi nei pori interni e attraverso di esso per raggiungere la maggior parte della proteina A) con caduta di alta pressione attraverso la colonna, il trasporto di massa negli adsorbenti a membrana è guidato dalla convezione alla rinfusa, con conseguente evitare limitazioni didiffusione 8,9,11,12. Inoltre, la caduta di pressione attraverso l'adsorbente a membrana è bassa, consentendo così portate di perfusione più elevate rispetto alle colonne imballate perline8,9,11,12. Pertanto, per l'isolamento su scala di laboratorio, si prevede che la cromatografia a membrana riduca i tempi di isolamento di diverse ore e aumenti la cattura del prodotto rispetto alla cromatografia a colonna. Inoltre, sono stati proposti modelli matematici per l'adsorbimento IgG negli adsorbenti a membrana8,9,11,12, consentendo così agli utenti finali di prevedere le prestazioni in laboratorio.
Un altro collo di bottiglia nei laboratori di scienze e ingegneria di base è la caratterizzazione della proteina che esprime fc. Tuttavia, la selezione di metodi appropriati per completare i test di caratterizzazione, come le macchie occidentali, può ridurre notevolmente il tempo dedicato ai test dei prodotti. Ad esempio, il trasferimento semidry in un sistema tampone discontinuo può realizzare il trasferimento elettroforetico di proteine da un gel SDS-PAGE a una membrana di polivinile difluoridina (PVDF) in pochi minuti, rispetto a 1-2 ore di trasferimento di serbatoio (bagnato) in un sistema tampone continuo13.
Un protocollo rapido, economico ed efficace per isolare e caratterizzare una proteina di fusione chimerica che esprime un frammento Fc di IgG umano è descritto nel presente documento. La maggior parte delle apparecchiature è prontamente disponibile nei laboratori standard di scienze biomediche di base, biologia, chimica e (bio)ingegneria chimica, e tutti i reagenti sono disponibili in commercio. Sebbene siano stati generati risultati rappresentativi dall'isolamento e dalla caratterizzazione di una proteina di fusione chimerica Fc murina-1/umana (Gal-1hFc), il protocollo semplificato può essere applicato all'isolamento di altre molecole che possiedono un frammento Fc, come anticorpi, frammenti Fc stessi o altre proteine di fusione Fc. I nostri miglioramenti hanno ridotto drasticamente i tempi di elaborazione (fino a 4 ore ma più in genere ~ 9 ore, rispetto a oltre 20 ore lavorative) ottenendo al contempo un recupero del prodotto simile o migliorato rispetto ai metodi standard.

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		<tr height="42">
			<td height="42" style="height:42px;width:313px;">Protein A membrane adsorber (Sartobind Protein A 2 ml)&#160;</td>
			<td style="width:191px;">Sartorius-Stedim</td>
			<td style="width:147px;">93PRAP06HB-12--A</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Amicon Ultra centrifugal filter, 10 kDa, 4 ml</td>
			<td style="width:191px;">Millipore</td>
			<td>UFC801008</td>
			<td>Use 15 ml volume if needed</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Sterile vacuum filter unit, 0.22 &#181;m, 500 ml</td>
			<td>Millipore</td>
			<td>SCGPU05RE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">10 ml Syringes with Luer Lock tips</td>
			<td>BD</td>
			<td>301604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">30 ml Syringes with&#160; Luer Lock tips</td>
			<td>BD</td>
			<td>309650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Tygon lab tubing (1/16 in x 1/8 in x 50 ft)</td>
			<td>Cole Parmer</td>
			<td>WU-95903-16</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:313px;">Slide-A-Lyzer MINI Dialysis Devices (10K MWCO)</td>
			<td>Thermo Scientific</td>
			<td>69576</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Snap i.d. antibody collection tray</td>
			<td>EMD Millipore</td>
			<td>WBAVDABTR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Snap i.d. single well blot holder</td>
			<td>EMD Millipore</td>
			<td>WBAVDBH01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Elution buffer</td>
			<td>Thermo Scientific</td>
			<td>21004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Tris, ultra pure</td>
			<td>Fisher Scientific</td>
			<td>819623</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Tween 20</td>
			<td>Fisher Scientific</td>
			<td>BP337-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">DPBS</td>
			<td>Thermo Scientific&#160;</td>
			<td>SH30028.02</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:313px;">DPBS with Ca2+/Mg2+</td>
			<td>Life Technologies</td>
			<td>14080-055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">BSA</td>
			<td>Sigma</td>
			<td>A9647</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Human Fc</td>
			<td>Bethyl Research Laboratories</td>
			<td>&#160;P80-104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Anti-human Fc-APC</td>
			<td>Jackson Immunoresearch</td>
			<td>109136170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Anti-human Fc-AP</td>
			<td>Bio-Rad</td>
			<td>170-5018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Alkaline phosphatase substrate</td>
			<td>Promega</td>
			<td style="width:147px;">S3841</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:313px;">Flow cytometry tubes (5 ml polystyrene or polypropylene)</td>
			<td>BD</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;"></td>
			<td style="width:191px;"></td>
			<td style="width:147px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Name of Material/ Equipment</td>
			<td style="width:191px;">Company</td>
			<td style="width:147px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>55-2226</td>
			<td style="width:173px;">A peristaltic pump is also acceptable</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Protein gel electrophoresis system&#160;</td>
			<td>Bio-Rad Laboratories</td>
			<td>552BR094876</td>
			<td style="width:173px;">Any gel electrophoresis system is acceptable, but mini gel systems run fastest</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;">Semidry blotter&#160;</td>
			<td>Bio-Rad Laboratories</td>
			<td style="width:147px;">690BR006163</td>
			<td style="width:173px;">Any transfer system is acceptable, but discontinuos transfer systems perform electrophoretic transfer fastest</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:313px;">SNAP i.d. vacuum-assisted protein detection system</td>
			<td style="width:191px;">EMD Millipore</td>
			<td>WBAVDBASE</td>
			<td style="width:173px;">An upgraded model has replaced this specific instrument, but either should work just as well</td>
		</tr>
	</tbody>
</table>

isolation and characterization of chimeric human fc-expressing proteins using protein a membrane adsorbers and a streamlined workflow

La purificazione su scala industriale su scala industriale delle biomolecole dai supernanti di coltura cellulare e dalle soluzioni cellulari liscivie può essere effettuata utilizzando la cromatografia di affinità. Mentre la cromatografia di affinità che utilizza proteine porose A agarose perline imballate in colonne è probabilmente il metodo più comune di isolamento in scala di laboratorio di anticorpi e proteine ricombinanti che esprimono frammenti fc di IgG, può essere un processo dispendioso in termini di tempo e costoso. I vincoli di tempo e finanziari sono particolarmente scoraggianti nei piccoli laboratori scientifici di base che devono recuperare centinaia di microgrammi a quantità di proteine milligrammi da soluzioni diluite, ma non hanno accesso a sistemi di erogazione di liquidi ad alta pressione e / o personale con esperienza in bioseparazioni. Inoltre, la quantificazione e la caratterizzazione dei prodotti possono anche allungare eccessivamente il tempo di lavorazione per diversi giorni lavorativi e gonfiare le spese (materiali di consumo, salari, ecc.). Pertanto, è necessario un protocollo veloce, economico ma efficace per l'isolamento su scala di laboratorio e la caratterizzazione di anticorpi e altre proteine in possesso di un frammento Fc. A tal fine, abbiamo ideato un protocollo che può essere completato da personale tecnico a esperienza limitata in meno di 9 ore (circa un giorno lavorativo) e fino a 4 ore, rispetto ai metodi tradizionali che richiedono oltre 20 ore lavorative. La maggior parte delle attrezzature richieste è prontamente disponibile nei laboratori standard di scienze biomediche, biochimiche e (bio)ingegneria chimica, e tutti i reagenti sono disponibili in commercio. Per dimostrare questo protocollo, vengono presentati risultati rappresentativi in cui la galettanina murina chimerica-1 fusa all'Fc umano (Gal-1hFc) dal supernatante di coltura cellulare è stata isolata usando una proteina A adsorbente di membrana. Il Gal-1hFc purificato è stato quantificato utilizzando una procedura accelerata di analisi western blotting e caratterizzato utilizzando citometria a flusso. Il flusso di lavoro semplificato può essere modificato per altre proteine che esprimono Fc, come gli anticorpi, e /o alterato per incorporare metodi alternativi di quantificazione e caratterizzazione.

Il protocollo semplificato per l'isolamento e la caratterizzazione delle proteine che esprimono fc viene regolarmente utilizzato per elaborare la galettanina murina chimerica-1 fusa all'Fc umano (Gal-1hFc) dai supernanti di coltura cellulare diluita. Il diagramma di flusso nella figura 2 illustra il flusso di lavoro e l'ora di ogni passaggio del protocollo. Per un lotto tipico di 300 ml di supernatante, il tempo totale di lavorazione è di circa 9 ore quando viene eseguita la prova citometrica del flusso...

Watch this Scientific Journal Video about Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow at JoVE.com

Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

Rispetto alla cromatografia di affinità tradizionale che utilizza colonne piene di perline di agarosio A, gli adsorbenti a membrana A proteici possono accelerare significativamente l'isolamento su scala di laboratorio degli anticorpi e di altre proteine che esprimono frammenti Fc. Metodi di analisi e quantificazione appropriati possono accelerare ulteriormente l'elaborazione delle proteine, consentendo di completare l'isolamento / caratterizzazione in un giorno lavorativo, anziché oltre 20 ore lavorative.

Isolamento e caratterizzazione delle proteine chimeriche che esprimono fc umano utilizzando proteine A adsorbenti di membrana e un flusso di lavoro semplificato

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using ...

isolation-characterization-chimeric-human-fc-expressing-proteins

Research

JoVE Journal

Bioengineering

7.1K Views.  Ohio University. Rispetto alla cromatografia di affinità tradizionale che utilizza colonne piene di perline di agarosio A, gli adsorbenti a membrana A proteici possono accelerare significativamente l'isolamento su scala di laboratorio degli anticorpi e di altre proteine che esprimono frammenti Fc. Metodi di analisi e quantificazione appropriati possono accelerare ulteriormente l'elaborazione delle proteine, consentendo di completare l'isolamento / caratterizzazione in un giorno lavorati...

Video: Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

La creazione di substrati Patterned bidimensionali per confinamento di proteine ​​e cellule

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Ricerca

Bioingegneria

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed secondary tumors1,2,3. CTCs are indicators of metastatic disease and their detection in blood samples may be used to diagnose cancer and monitor a patient&prime;s response to therapy. Since CTCs are rare, comprising about one tumor cell among billions of normal blood cells in advanced cancer patients, their detection and enumeration is a difficult task. We exploit the presence of pigment in most melanoma cells to generate photoacoustic, or laser induced ultrasonic waves in a custom flow cytometer for detection of circulating melanoma cells (CMCs)4,5. This process entails separating a whole blood sample using centrifugation and obtaining the white blood cell layer. If present in whole blood, CMCs will separate with the white blood cells due to similar density. These cells are resuspended in phosphate buffered saline (PBS) and introduced into the flowmeter. Rather than a continuous flow of the blood cell suspension, we induced two phase flow in order to capture these cells for further study. In two phase flow, two immiscible liquids in a microfluidic system meet at a junction and form alternating slugs of liquid6,7. PBS suspended white blood cells and air form microliter slugs that are sequentially irradiated with laser light. The addition of a surfactant to the liquid phase allows uniform slug formation and the user can create different sized slugs by altering the flow rates of the two phases. Slugs of air and slugs of PBS with white blood cells contain no light absorbers and hence, do not produce photoacoustic waves. However, slugs of white blood cells that contain even single CMCs absorb laser light and produce high frequency acoustic waves. These slugs that generate photoacoustic waves are sequestered and collected for cytochemical staining for verification of CMCs.

We have developed a flow cytometer using laser induced ultrasound to detect circulating melanoma cells as an early indicator of metastatic disease.

Rilevazione e isolamento delle cellule del melanoma circolanti con flussimetria fotoacustico

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to identify and characterize their interactions are necessary5. To this end, we developed the Membrane Strep-protein interaction experiment, called Membrane-SPINE6. This technique combines in vivo cross-linking using the reversible cross-linker formaldehyde with affinity purification of a Strep-tagged membrane bait protein. During the procedure, cross-linked prey proteins are co-purified with the membrane bait protein and subsequently separated by boiling. Hence, two major tasks can be executed when analyzing protein-protein interactions (PPIs) of membrane proteins using Membrane-SPINE: first, the confirmation of a proposed interaction partner by immunoblotting, and second, the identification of new interaction partners by mass spectrometry analysis. Moreover, even low affinity, transient PPIs are detectable by this technique. Finally, Membrane-SPINE is adaptable to almost any cell type, making it applicable as a powerful screening tool to identify PPIs of membrane proteins.

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

A biochemical approach is described to identify in vivo protein-protein interactions (PPI) of membrane proteins. The method combines protein cross-linking, affinity purification and mass spectrometry, and is adaptable to almost any cell type or organism. With this approach, even the identification of transient PPIs becomes possible.

Membrana-SPINE: uno strumento biochimici per identificare interazioni proteina-proteina di proteine ​​di membrana<em&gt; In Vivo</em

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to ...

Synthesis of an Intein-mediated Artificial Protein 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 this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

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 ...

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.

We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.

Caratterizzazione dei sistemi complessi Utilizzando il Design of Experiments Approccio: Transient Protein Expression in tabacco come un caso di studio

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing&#160; a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

Developing biotinylatable fusion proteins has many potential applications in various fields of research. Recombinant protein engineering is a straight forward procedure that is cost-effective, providing high yields of custom-designed proteins.

Espressione, isolamento e purificazione di solubili e insolubili Biotinylated proteine ​​per Nerve Tissue Regeneration

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

Luminescence Resonance Energy Transfer allo Studio conformazionale Cambiamenti nella membrana proteine ​​espresse in cellule di mammifero

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

Produzione, caratterizzazione e possibilità di utilizzo di un 3D tissutale umano esofageo mucose Modello

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should mimic the human tissues they are aiming to replace; to provide the required anatomical shape, the materials must be able to sustain the mechanical forces they will experience when implanted at the defect site. Although the mechanical properties of tissue-engineered scaffolds are of great importance, many human tissues that undergo restoration with engineered materials have not been fully biomechanically characterized. Several compressive and tensile protocols are reported for evaluating materials, but with large variability it is difficult to compare results between studies. Further complicating the studies is the often destructive nature of mechanical testing. Whilst an understanding of tissue failure is important, it is also important to have knowledge of the elastic and viscoelastic properties under more physiological loading conditions.
This report aims to provide a minimally destructive protocol to evaluate the compressive and tensile properties of human soft tissues. As examples of this technique, the tensile testing of skin and the compressive testing of cartilage are described. These protocols can also be directly applied to synthetic materials to ensure that the mechanical properties are similar to the native tissue. Protocols to assess the mechanical properties of human native tissue will allow a benchmark by which to create suitable tissue-engineered substitutes.

Tissue biomechanics is important for maintaining cell shape and function and for determining phenotype. This report demonstrates non-destructive mechanical protocols for characterizing elastic and viscoelastic properties of human soft tissues, which can be directly applied to tissue-engineered substrates to allow a close matching of engineered materials to native tissue.

Caratterizzazione biomeccanica di morbidi tessuti umani usando una indentazione e prove di trazione

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

Fabbricazione e caratterizzazione dei fantasmi di tessuto ottico contenente macrostruttura

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...