1. Costruzione plasmide <ol><li> Costruire un plasmide di espressione (pBAD-HerFab-L177TAG) che esprimesse il gene bersaglio degli anticorpi (pBAD-HerFab-WT) con il suo 6 -tag, e sostituire il codone per la leucina-177 con il codone ambra (TAG) 27, utilizzando convenzionale tecnica di mutagenesi sito-diretta. Vedere la Tabella dei Materiali. </li><li> Costruire un altro plasmide di espressione (pEVOL-AFRS) contenente i geni per il tRNA evoluta Tyr e-tRNA sintetasi coppia (AARS). Utilizzare il vettore plasmide appositamente progettato, pEVOL, per un efficiente incorporazione di UAAs. Le dettagliate protocolli di informazione plasmide e la clonazione sono descritti nella relazione precedente. 28 </li><li> Ottenere DNA plasmidico di alta qualità utilizzando kit di preparazione plasmide disponibili in commercio. Il rapporto di 260/280 di ~ 1.8 è ottimale per il DNA purificato. Se richiesto, eseguire 1% elettroforesi su gel di agarosio per verificare la purezza del DNA. </li></ol><ol><li> elettroporazione <ol><li> Aggiungere ogni DNA 1 ml di pEVOL-AFRS 28 e pBAD-HerFab-L177TAG a 20 microlitri ceppo di Escherichia coli DH10β. Mescolare delicatamente, con una pipetta. I plasmidi possono essere trasformati insieme o in reazioni indipendenti. </li><li> Per 0,1 cm cuvette, configurare le impostazioni di elettroporazione a 25 mF e 2,5 kV. </li><li> Inserire la cuvetta nella diapositiva della camera di scioccante. Spingere la slitta nella camera fino a quando la provetta entra in contatto costante con gli elettrodi da camera. </li><li> Pulse una volta premendo il pulsante di impulso fino a quando il segnale acustico della macchina elettroporazione. </li><li> Aggiungere 1 ml di brodo eccellente ottimale con catabolite rimozione (SOC) mezzo contenente 2% (w / v) triptone, 0,5% (w / v) di estratto di lievito, 10 mM NaCl, 2,5 mM KCl, 10 mM MgCl 2, e 20 mM di glucosio alla cuvetta rapidamente e trasferire il composto in una provetta. Incubare le cellule per 1 ora a 37 ° C con agitazione.</li><li> Le cellule di E. coli diffusione trasformato in lisogenia brodo (LB) piastre di agar contenenti antibiotici appropriati (35 mg / ml cloramfenicolo e 100 mg / ml ampicillina per la selezione pEVOL-AFRS e pBAD-HerFab-L177TAG, rispettivamente). Incubare le piastre a 37 ° C per 12 h. </li></ol></li><li> preparazione Cultura <ol><li> Inoculare una singola colonia trasformata in 5 ml LB medie contenenti antibiotici. Incubare per 12 ore a 37 ° C con agitazione. </li></ol></li></ol> 3. Espressione e purificazione di HerFab-L177AF <ol><li> L&#39;espressione di HerFab-L177AF <ol><li> Trasferire la coltura primaria (5 mL) in 200 mL LB contenente ampicillina media (100 ug / mL) e AF (1 mM), seguita da incubazione a 37 ° C con agitazione. NOTA: soluzione madre 100 mM AF (10 mL) è stata preparata sciogliendo 206 mg AF in 100 mM di acido cloridrico (10 mL, volume finale). </li><li> Aggiungere 2 ml di 1,6 M arabinosio (16mM concentrazione finale) quando la cultura raggiunge il log-fase (OD 550 = 0,8, OD = densità ottica). Incubare per 12 ore a 30 ° C con agitazione. </li><li> Celle di raccolta per centrifugazione a 11.000 xg per 5 min. Gettare il surnatante e congelare il pellet a -20 ° C. </li></ol></li><li> La lisi cellulare <ol><li> Risospendere il pellet di cellule in 20 mL di tampone di lisi periplasmic contenente 30 mM Tris (pH 8,0), 1 mM EDTA, 20% di saccarosio e 0,2 mg / ml di lisozima e incubare la miscela per 1 ora a 37 ° C. </li><li> Centrifugare il lisato cellulare a 18.000 xga 4 ° C per 15 min. Trasferire il surnatante in una nuova provetta e scartare il pellet. </li></ol></li><li> Ni-NTA cromatografia di affinità <ol><li> Aggiungere sospensione resina Ni-NTA ad ogni provetta da centrifuga (400 microlitri di resina per una cultura 200 mL), e mescolare delicatamente a 4 ° C per 1 h. </li><li> Versare la sospensione in una colonna polipropilene e lavare la resina tre volte con 5 mL bu lavaggioffer contenente 50 mM NaH 2 PO 4 (pH 8,0), 20 mM imidazolo, e 300 mm NaCl. </li><li> Eluire l&#39;anticorpo bersaglio con 300 microlitri di tampone di eluizione contenente 50 mM NaH 2 PO 4 (pH 8,0), 250 mm imidazolo, e 300 mm NaCl. </li><li> Determinare la concentrazione della proteina mutante Metodo di Bradford 29 o misurando l&#39;assorbanza a 280 nm. Calcolare il coefficiente di estinzione (75.866 M -1 cm -1) per HerFab-L177AF a 280 nm da proteine estinzione calcolatrice coefficiente utilizzando il coefficiente di estinzione (2.471 M -1 cm -1) per AF. NOTA: La sequenza aminoacidica del HerFab può essere trovata sul sito NCBI (GI: 783.282.791 e GI: 783.282.792). </li></ol></li></ol> 4. Coniugazione purificata HerFab-L177AF con Acetilene sonde Utilizzando cicloaddizione azide-alchino Strain-promossa (SPAAC) <ol><li> Aggiungere 20 ml di Cy5.5-Azadibenzocyclooctyne (Cy5.5-ADIBO) in H 2 O (20081; M concentrazione finale) ad una soluzione di 10 ml di HerFab-L177AF (10 mM concentrazione finale) in tampone fosfato contenente 10 mM Na 2 HPO 4 (pH 7,0) e 100 mM NaCl. NOTA: Come alternative, cyclooctyne difluorinated (DIFO) e biciclo [6.1.0] nonyne (BCN) derivati ​​sono disponibili per la stessa applicazione anche. </li><li> Lasciare che la reazione di cicloaddizione strain-promosso procedere per 6 ore a 37 ° C. Se viene utilizzata una sonda fotosensibile (ad esempio, Cy5.5), coprire il recipiente di reazione con un foglio di alluminio. </li><li> Purificare il HerFab etichettati in base al punto 5. </li></ol> 5. Purificazione di etichetta HerFab <ol><li> Aggiungere 500 ml di campione in una colonna di filtro rotativo centrifugo. </li><li> Centrifugare la colonna di spin a 14000 g a 4 ° C per 15 min. </li><li> Gettare flusso continuo e il trasferimento purificati campione dalla colonna di selezione per una provetta da microcentrifuga da 1,5. </li><li> In alternativa al passo 5.1 5.3, eseguire purificazione mediante dialisi contro lo stesso tampone. </li><li> Conservare il purificato etichettato HerFab a 4 ° C. </li></ol> 6. SDS-PAGE analisi di etichetta HerFab <ol><li> Aggiungere 5 ml LDS tampone proteica contenente 106 mm Tris · HCl, 141 mm di base Tris (pH 8,5), 2% LDS, 10% glicerolo, 0,51 mm EDTA, di 0,22 mm SERVA Blu G250, e 0,175 mm Rosso fenolo a 13 ml di purificato etichettati HerFab (7.8 mM o 0,38 mg / ml) in presenza (+) o in assenza (-) di 2 microlitri ditiotreitolo (DTT, 100 mM concentrazione finale). Incubare la miscela a 95 ° C per 10 min. </li><li> Fissare il 4-12% Bis-Tris gel SDS-PAGE cassette alla cella di elettroforesi e aggiungere tampone di corsa. Caricare i campioni di proteine ​​coniugate e il marcatore di peso molecolare pre-colorato. Eseguire l&#39;elettroforesi su gel per 35 minuti a 200 V. Nota: Mantenere la cella di elettroforesi al buio durante l&#39;intero periodo per ridurre al minimo fotometabolismo del fluoroforo. </li><li> Dopo l&#39;elettroforesi, trasferireil gel ad uno scanner gel fluorescenti, e scansione per l&#39;emissione di fluorescenza alla lunghezza d&#39;onda appropriata. Per Cy5.5, utilizzare la modalità Cy5 nel software dello scanner. </li><li> Macchia il gel con una macchia proteine ​​commerciale. </li></ol>

<ol>
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The authors have nothing to disclose.

L&#39;incorporazione genetica di aminoacidi non naturali in proteine ​​ha diversi vantaggi rispetto ad altri metodi utilizzati per la modifica della proteina. 1-3 Uno dei vantaggi importanti è la sua applicabilità generale a qualsiasi tipo di proteina. In linea di principio, non vi è alcuna limitazione nella scelta di una proteina bersaglio e un sito target della proteina. Tuttavia, la sostituzione di un residuo strutturalmente o funzionalmente importante con SAU può comportare alterando la struttura ...

Dal momento che la costituzione genetica di p -methoxyphenylalanine in Escherichia coli è stato segnalato, 1 più di 100 amminoacidi non naturali (UAAs) sono stati inseriti con successo in varie proteine. 1-3 Tra questi UAAs, gli aminoacidi contenenti gruppi funzionali bioorthogonal sono stati ampiamente studiati e rappresentano la percentuale più elevata. I gruppi funzionali bioorthogonal utilizzati nei UAAs includono chetone, 4 azide, 5 alchino, 6 cyclooctyne, 7 tetrazina, 8 α, β-insaturo ammide, 9 norbonene, 10 transcyclooctene, 11 e biciclo [6.1.0] -nonyne. 11 Sebbene ogni gruppo funzionale ha i suoi vantaggi e svantaggi, gli amminoacidi azide contenenti più sono stati ampiamente utilizzati per la coniugazione di proteine. p -Azidophenylalanine (AF), uno degli amminoacidi azido-contenente, è prontamente disponibile, e la sua incorporazione efficiency è eccellente. proteine ​​mutanti contenenti questo aminoacido può essere fatto reagire con alchini di cicloaddizione di rame-catalizzata o con cyclooctynes ​​di SPAAC. 12-20 Recentemente, biofarmaceutici hanno attratto grande attenzione nel settore farmaceutico. Il coniugato anticorpo-farmaco (ADC) è una classe di anticorpi terapeutici che sono vantaggiosi per la loro capacità di terapia mirata per il trattamento dei tumori umani 21 e altre malattie. Più di 50 ADC sono attualmente in sperimentazione clinica, e il numero è in rapido aumento. Nello sviluppo di ADC, molti fattori devono essere considerati per massimizzare l&#39;efficacia e minimizzare gli effetti collaterali. Tra questi fattori, una reazione di coniugazione efficiente e sito-specifica per formare un legame covalente tra un anticorpo ed un farmaco è critica. L&#39;efficienza desiderata e specificità nella reazione di coniugazione può essere ottenuto mediante coniugazione con un gruppo funzionale in un bioorthogonalamminoacido non naturale che è specificamente incorporata in un anticorpo. 22-26 Qui, si segnala un protocollo al sito-specifico incorporano AF in un frammento di anticorpo e coniugare il frammento di anticorpo mutante con una sonda biochimico.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1. plasmid Construction</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>plasmid pBAD_HerFab_L177TAG</td>
			<td></td>
			<td></td>
			<td>optionally contain the amber stop codon(TAG) at a desired position. Ko, W. et al. Efficient and Site-Specific Antibody Labeling by Strain-promoted Azide-Alkyne Cycloaddition. BKCS. 36 (9), 2352-2354, doi: 10.1002/bkcs.10423, (2015)</td>
		</tr>
		<tr>
			<td>plasmid pEvol-AFRS</td>
			<td></td>
			<td></td>
			<td>Young, T. S., Ahmad, I., Yin, J. A., and Schultz, P. G. An enhanced system for unnatural amino acid mutagenesis in E. coli. J. Mol. Biol. 395 (2), 361-374, doi: 10.1016/j.jmb.2009.10.030, (2010)</td>
		</tr>
		<tr>
			<td>DH10B</td>
			<td>Invitrogen</td>
			<td>C6400-03</td>
			<td>Expression Host</td>
		</tr>
		<tr>
			<td>Plasmid Mini-prep kit</td>
			<td>Nucleogen</td>
			<td>5112</td>
			<td>200/pack</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Intron biotechnology</td>
			<td>32034</td>
			<td>500g</td>
		</tr>
		<tr>
			<td>Ethidium bromide</td>
			<td>Alfa Aesar</td>
			<td>L07482</td>
			<td>1g</td>
		</tr>
		<tr>
			<td>LB Broth</td>
			<td>BD Difco</td>
			<td>244620</td>
			<td>500g</td>
		</tr>
		<tr>
			<td>2. Culture preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2.1) Electroporation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro pulser&#160;</td>
			<td>BIO-RAD</td>
			<td>165-2100</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro pulser cuvette</td>
			<td>BIO-RAD</td>
			<td>165-2089</td>
			<td>0.1cm electrode gap, pkg. of 50</td>
		</tr>
		<tr>
			<td>Ampicillin Sodium</td>
			<td>Wako</td>
			<td>018-10372</td>
			<td>25g</td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Alfa Aesar</td>
			<td>B20841</td>
			<td>25g</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>SAMCHUN</td>
			<td>214230</td>
			<td>500g</td>
		</tr>
		<tr>
			<td>SOC medium</td>
			<td>Sigma</td>
			<td>S1797</td>
			<td>100ML</td>
		</tr>
		<tr>
			<td>3. Expression and purification of HerFab-L177AF</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3.1 Expression of Herfab-L177AF</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>p-azido-L-phenylalanine (AF)</td>
			<td>Bachem</td>
			<td>F-3075.0001</td>
			<td>1g</td>
		</tr>
		<tr>
			<td>L(+)-Arabinose, 99%</td>
			<td>Acros</td>
			<td>104981000</td>
			<td>100g</td>
		</tr>
		<tr>
			<td>Hydrochloric acid, 35~37%</td>
			<td>SAMCHUN</td>
			<td>H0256</td>
			<td>500ml</td>
		</tr>
		<tr>
			<td>3.2 Cell lysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)aminomethane, 99%</td>
			<td>SAMCHUN</td>
			<td>T1351</td>
			<td>500g</td>
		</tr>
		<tr>
			<td>EDTA disodium salt dihydrate, 99.5%</td>
			<td>SAMCHUN</td>
			<td>E0064</td>
			<td>1kg</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S9378</td>
			<td>500g</td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Siyaku</td>
			<td>126-0671</td>
			<td>1g</td>
		</tr>
		<tr>
			<td>3.3 Ni-NTA Affinity Chromatography</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA resin</td>
			<td>QIAGEN</td>
			<td>30210</td>
			<td>25ml</td>
		</tr>
		<tr>
			<td>Polypropylene column</td>
			<td>QIAGEN</td>
			<td>34924</td>
			<td>50/pack, 1ml capacity</td>
		</tr>
		<tr>
			<td>Imidazole, 99%</td>
			<td>SAMCHUN</td>
			<td>I0578</td>
			<td>1kg</td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic, 98%</td>
			<td>SAMCHUN</td>
			<td>S0919</td>
			<td>1kg</td>
		</tr>
		<tr>
			<td>Sodium Chloride, 99%</td>
			<td>SAMCHUN</td>
			<td>S2907</td>
			<td>1kg</td>
		</tr>
		<tr>
			<td>4. Conjugation of Purified HerFab-L177AF with Alkyne Probes Using Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC)&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cy5.5-ADIBO&#160;</td>
			<td>FutureChem</td>
			<td>FC-6119</td>
			<td>1mg</td>
		</tr>
		<tr>
			<td>5. Purification of Labeled HerFab</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra 0.5 mL Centrifugal Filters</td>
			<td>MILLIPORE</td>
			<td>UFC500396</td>
			<td>96/pack, 500ul capacity</td>
		</tr>
		<tr>
			<td>6. SDS-PAGE Analysis of Labeled HerFab and Fluorescent Gel Scanning</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1,4-Dithio-DL-threitol, DTT, 99.5 %</td>
			<td>Sigma</td>
			<td>10708984001</td>
			<td>10g</td>
		</tr>
		<tr>
			<td>NuPAGE LDS Sample Buffer, 4X</td>
			<td>Thermofisher</td>
			<td>NP0007</td>
			<td>10ml</td>
		</tr>
		<tr>
			<td>MES running buffer</td>
			<td>Thermofisher</td>
			<td>NP0002</td>
			<td>500ml</td>
		</tr>
		<tr>
			<td>Nupage Novex 4-12% SDS PAGE gels</td>
			<td>Thermofisher</td>
			<td>NO0321</td>
			<td>12well</td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue R-250</td>
			<td>Wako</td>
			<td>031-17922</td>
			<td>25g</td>
		</tr>
		<tr>
			<td>Typhoon 9210 variable mode imager</td>
			<td>Amersham Biosciences</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

efficient and site-specific antibody labeling by strain-promoted azide-alkyne cycloaddition

Ci sono attualmente molti strumenti chimici a disposizione per introdurre sonde chimiche in proteine ​​per studiare la loro struttura e funzione. Un metodo utile è la coniugazione di proteine ​​geneticamente introducendo un amminoacido non naturale contenente un gruppo funzionale bioorthogonal. Questo rapporto descrive un protocollo dettagliato per site-specific anticorpi coniugazione. Il protocollo comprende dettagli sperimentali per la costituzione genetica di un aminoacido azide contenenti, e la reazione di coniugazione per cicloaddizione azide-alchino strain-promosso (SPAAC). Questa reazione strain-promosso procede per semplice miscelazione delle molecole reagenti a pH fisiologico e temperatura, e non richiede reagenti aggiuntivi come rame (I) ioni e leganti rame-chelanti. Pertanto, questo metodo sarebbe utile per la coniugazione di proteine ​​generale e sviluppo di anticorpi coniugati farmaco (ADC).

<html> In questo studio, un frammento di anticorpo era sito-specifico coniugato con un fluorocromo incorporando un aminoacido azide contenente nel frammento e reagire il frammento di anticorpo mutante con un cyclooctyne teso (Figura 1). HerFab è stato selezionato come il frammento di anticorpo di destinazione in cui AF è stato accolto come un aminoacido azide contenenti. Per scegliere il residuo in HerFab per la sostituzione con AF, la struttura cristallina ai raggi X d...

Watch this Scientific Journal Video about Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition at JoVE.com

Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition

Here, we present a protocol to site-specifically introduce chemical probes into an antibody fragment by genetically incorporating an azide-containing amino acid, and subsequently coupling the azide with a chemical probe by strain-promoted azide-alkyne cycloaddition (SPAAC).

Efficiente e anticorpi etichettatura per cicloaddizione azide-alchino Strain-promosso site-specific

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is ...

efficient-site-specific-antibody-labeling-strain-promoted-azide

Research

JoVE Journal

Biochemistry

20.5K Views.  Sogang University. Here, we present a protocol to site-specifically introduce chemical probes into an antibody fragment by genetically incorporating an azide-containing amino acid, and subsequently coupling the azide with a chemical probe by strain-promoted azide-alkyne cycloaddition (SPAAC).

Video: Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several target identification methods have been developed and used to successfully elucidate the binding proteins of a variety of small molecules, these techniques have drawbacks that make them unsuitable for detecting certain types of small molecule-target interactions. In particular, non-covalent interactions that depend on native cellular conditions, such as those of membrane proteins whose structures may be perturbed upon cell lysis, are often not amenable to affinity-based target identification methods. Here, we demonstrate a method wherein a probe containing a photolabile group is used to covalently crosslink to the small molecule binding protein within the environment of the live cell, allowing the detection and isolation of the target protein without the need for maintenance of the interaction after cell lysis. This technique is a valuable tool for studying biologically interesting small molecules with unknown mechanisms, both in the context of basic biology as well as drug discovery.

We describe here a method for identification of small molecule-binding proteins using photoaffinity labeling. The advantage of this technique is that binding and covalent labeling of the target proteins occurs within the live cellular environment, removing the risk of disrupting native protein structure and binding conditions upon cell lysis.

L&#39;identificazione di piccole proteine ​​che legano molecole in un nativo Cellular ambiente da Live-cell Photoaffinity Etichettatura

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several ...

Ricerca

Biochimica

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. Improving drug delivery to maximize therapeutic outcomes and to reduce drug-associated side effects are some of the cornerstones of present-day nanomedicine. Nanoparticles in particular have found a wide application in cancer treatment. Nanoparticles that offer a high degree of flexibility in design, application, and production based on the tumor microenvironment are projected to be more effective with rapid translation into clinical practice. The polymeric micellar nano-carrier is a popular choice for drug delivery applications.
In this article, we describe a simple and effective protocol for synthesizing drug-loaded, disulfide cross-linked micelles based on the self-assembly of a well-defined amphiphilic linear-dendritic copolymer (telodendrimer, TD). TD is composed of polyethylene glycol (PEG) as the hydrophilic segment and a thiolated cholic acid cluster as the core-forming hydrophobic moiety attached stepwise to an amine-terminated PEG using solution-based peptide chemistry. Chemotherapy drugs, such as paclitaxel (PTX), can be loaded using a standard solvent evaporation method. The O2-mediated oxidation was previously utilized to form intra-micellar disulfide cross-links from free thiol groups on the TDs. However, the reaction was slow and not feasible for large-scale production. Recently, an H2O2-mediated oxidation method was explored as a more feasible and efficient approach, and it was 96 times faster than the previously reported method. Using this approach, 50 g of PTX-loaded, disulfide cross-linked nanoparticles have been successfully produced with narrow particle size distribution and high drug loading efficiency. The stability of the resulting micelle solution is analyzed using disrupting conditions such as co-incubation with a detergent, sodium dodecyl sulfate, with or without a reducing agent. The drug-loaded, disulfide cross-linked micelles demonstrated less hemolytic activity when compared to their non-cross-linked counterparts.

To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

Un approccio Facile ed efficiente per la produzione di micelle reversibile Disulfide Croce-linked

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

Laboratory Scale Production and Purification of a Therapeutic Antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include preparation of vector DNA, stable transfection and serum-free adaptation of a human embryonic kidney 293 cell line, set up of large scale cultures and purification using affinity chromatography.

Laboratorio scala di produzione e purificazione di un anticorpo terapeutico

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

There is an increasing demand to analyze many biological samples for disease understanding and biomarker discovery. Quantitative proteomics strategies that allow simultaneous measurement of multiple samples have become widespread and greatly reduce experimental costs and times. Our laboratory developed a technique called combined precursor isotopic labeling and isobaric tagging (cPILOT), which enhances sample multiplexing of traditional isotopic labeling or isobaric tagging approaches. Global cPILOT can be applied to samples originating from cells, tissues, bodily fluids, or whole organisms and gives information on relative protein abundances across different sample conditions. cPILOT works by 1) using low pH buffer conditions to selectively dimethylate peptide N-termini and 2) using high pH buffer conditions to label primary amines of lysine residues with commercially-available isobaric reagents (see Table of Materials/Reagents). The degree of sample multiplexing available is dependent on the number of precursor labels used and the isobaric tagging reagent. Here, we present a 12-plex analysis using light and heavy dimethylation combined with six-plex isobaric reagents to analyze 12 samples from mouse tissues in a single analysis. Enhanced multiplexing is helpful for reducing experimental time and cost and more importantly, allowing comparison across many sample conditions (biological replicates, disease stage, drug treatments, genotypes, or longitudinal time-points) with less experimental bias and error. In this work, the global cPILOT approach is used to analyze brain, heart, and liver tissues across biological replicates from an Alzheimer&#39;s disease mouse model and wild-type controls. Global cPILOT can be applied to study other biological processes and adapted to increase sample multiplexing to greater than 20 samples.

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT

Combined precursor isotopic labeling and isobaric tagging (cPILOT) is a quantitative proteomics strategy that enhances sample multiplexing capabilities of isobaric tags. This protocol describes the application of cPILOT to tissues from an Alzheimer's disease mouse model and wild-type controls.

Maggiore Campione Multiplexing dei tessuti utilizzando combinata Precursore isotopica etichettatura e isobarica Tagging (CPilot)

There is an increasing demand to analyze many biological samples for disease understanding and biomarker discovery. Quantitative proteomics strategies ...

Analysis of Histone Antibody Specificity with Peptide Microarrays

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The mass production and distribution of antibodies specific to histone PTMs has greatly facilitated research on these marks. As histone PTM antibodies are key reagents for many chromatin biochemistry applications, rigorous analysis of antibody specificity is necessary for accurate data interpretation and continued progress in the field. This protocol describes an integrated pipeline for the design, fabrication and use of peptide microarrays for profiling the specificity of histone antibodies. The design and analysis aspects of this procedure are facilitated by ArrayNinja, an open-source and interactive software package we recently developed to streamline the customization of microarray print formats. This pipeline has been used to screen a large number of commercially available and widely used histone PTM antibodies, and data generated from these experiments are freely available through an online and expanding Histone Antibody Specificity Database. Beyond histones, the general methodology described herein can be applied broadly to the analysis of PTM-specific antibodies.

This manuscript describes methods for applying peptide microarray technology to specificity profiling of antibodies that recognize histones and their post-translational modifications.

Analisi della specificità degli anticorpi istonici con microarray di peptide

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The ...

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

Proteoma profonda profilatura di etichettatura isobarico, vasto cromatografia liquida, spettrometria di massa e quantificazione assistita da Software

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

Metodo per ripiegamento efficiente e purificazione del chemocettore Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and podocytes with their slit diaphragms. The delicate structure of the glomerular filter, especially the slit diaphragm, relies on the interplay of diverse cell surface proteins. Studying these cell surface proteins has so far been limited to in vitro studies or histologic analysis. Here, we present a murine in vivo biotinylation labeling method, which enables the study of glomerular cell surface proteins under physiologic and pathophysiologic conditions. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of a protein of interest. Semi-quantitation of glomerular cell surface abundance is readily available with this novel method, and all proteins accessible to biotin perfusion and immunoprecipitation can be studied. In addition, isolation of glomeruli with or without biotinylation enables further analysis of glomerular RNA and protein as well as primary glomerular cell culture (i.e., primary podocyte cell culture).

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Here we present a protocol for murine in vivo labeling of glomerular cell surface proteins with biotin. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of the protein of interest.

Isolamento dei Glomeruli e In Vivo di etichettatura delle proteine di superficie delle cellule glomerulari

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

Quantificazione di tutto il proteoma di etichettatura omogeneità a livello di singola molecola

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the dissection and manipulation of biological pathways. Currently, only a limited number of chemically induced dimerization (CID) systems exist and engineering new ones with desired sensitivity and selectivity for specific small-molecule ligands remains a challenge in the field of protein engineering. We here describe a high throughput screening method, combinatorial binders-enabled selection of CID (COMBINES-CID), for the de novo engineering of CID systems applicable to a large variety of ligands. This method uses the two-step selection of a phage-displayed combinatorial nanobody library to obtain 1) &#34;anchor binders&#34; that first bind to a ligand of interest and then 2) &#34;dimerization binders&#34; that only bind to anchor binder-ligand complexes. To select anchor binders, a combinatorial library of over 109 complementarity-determining region (CDR)-randomized nanobodies is screened with a biotinylated ligand and hits are validated with the unlabeled ligand by bio-layer interferometry (BLI). To obtain dimerization binders, the nanobody library is screened with anchor binder-ligand complexes as targets for positive screening and the unbound anchor binders for negative screening. COMBINES-CID is broadly applicable to select CID binders with other immunoglobulin, non-immunoglobulin, or computationally designed scaffolds to create biosensors for in vitro and in vivo detection of drugs, metabolites, signaling molecules, etc.

Creating chemically induced protein dimerization systems with desired affinity and specificity for any given small molecule ligand would have many biological sensing and actuation applications. Here, we describe an efficient, generalizable method for de novo engineering of chemically induced dimerization systems via the stepwise selection of a phage-displayed combinatorial single-domain antibody library.

Creazione di sistemi di dimerizzazione delle proteine altamente specifici indotta chimicamente mediante la selezione stepwise di phage di una biblioteca combinatoria di anticorpi a dominio singolo