Name: isolation and characterization of chimeric human fc-expressing proteins using protein a membrane adsorbers and a streamlined workflow
Uploaded: 2014-01-08T14:15:00
Description: 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

Forfatterne ønsker at takke Mr. Luis F. Delgadillo (Institut for Kemisk og Biomolekylær Engineering, Ohio University), Matthew H. Williams (Institut for Kemisk og Biomolekylær Engineering, Ohio University), og Dr. Filiberto Cedeno-Laurent (Brigham og Women's Hospital og Harvard Medical School) for ekspert teknisk bistand. Forfatterne vil også gerne takke Winter 2011 CHE 404/BME 504 og Fall 2012 CHE 4830/BME 5830 studerende (Institut for Kemisk og Biomolekylær Engineering og Biomedicinsk Engineering Program, Ohio University) for teknisk rådgivning og diskussion. Dette arbejde blev støttet af National Science Foundation (NSF) Major Research Instrumentation grant CBET-1039869 (MMB), NSF CBET-1106118 (MMB), Dermatology Foundation Research Grant A050422 (SRB), National Institutes of Health (NIH) NCI tilskud 1R15CA16 1830-01 (MMB), NIH Kirschstein-NRSA Postdoctoral Fellowship F32CA144219-01A1 (SRB), NIH/NCI R01CA173610 (CJD) og NIH NCCAM tilskud R01AT004628 (CJD).

1. Kontroller affiniteten for protein A
<ol><li>Kontroller, at det Fc-udtrykkende protein (rekombinant fusionsprotein eller IgG-antistof, herunder benævnt "protein") har acceptabel affinitet for protein A14-17, inden proteinet A-membrane adsorber anvendes, og test for tilstedeværelsen af proteinet i stamopløsningen(f.eks. cellekulturens supernatant afklaret ved centrifugering ved 1.000 x g i 5 min. til pellet suspenderede celler). Brug flowcytometri, vestlig blotting, ELISA osv. at opdag...

<ol>
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Protokollen beskrevet heri blev udviklet til hurtigt at isolere og karakterisere et kimært fusionsprotein, der udtrykker menneskelige Fc (Gal-1hFc), uden at det væsentligt kompromitterer produktgenvinding eller oppustningsomkostninger. Nøglekomponenterne i den strømlinede arbejdsgang er et protein A-membran adsorber til isolation, og halvdryk overførsel og vakuumassisteret immunoblotting til karakterisering af vestlig blotting.
Det dramatiske fald i behandlingstiden for isolation og kara...

Isolering af antistoffer og rekombinante proteiner, der udtrykker immunglobulin G (IgG) Fc fragmenter fra cellekultur supernatants og fortyndet lyserede celleopløsninger kan opnås ved hjælp af protein A affinitet kromatografi. I grundlæggende videnskabs- og ingeniørlaboratorier og industri anvendes kolonner fyldt med porøst protein A-belagt agarose, glas eller polymere perler oftest til affinitetskromatografi på trods af høje økonomiske omkostninger og lange behandlingstider1-3. Det er godt værdsat, at affinitetskromatografi repræsenterer den største udgift og behandling af flaskehals i både laboratorie- og industrimiljøer2,4. Som følge heraf er der udviklet en lang række forbedringer for at reducere omkostninger og behandlingstid uden at ofre den samlede produktgenvinding fra procestoget1,2,5-7. Særligt lovende for isolering af antistoffer og andre Fc-udtrykke proteiner er affinitet kromatografi ved hjælp af et protein A membran adsorber2,5,7-10. Mens Fc indfangning i kolonnekromatografi er diffusionsbegrænset (dvs. at det fc-udtrykkende protein skal spredes ind i og gennem indvendige porer for at nå størstedelen af proteinet A) med højt trykfald over kolonnen, er massetransport i membran adsorbere drevet af bulk konvektion, hvilket resulterer i undgåelse af diffusionsbegrænsninger8,9,11,12. Derudover er trykfaldet på tværs af membranens adsorber lavt, hvilket muliggør hurtigere perfusionsflowhastigheder sammenlignet med perlepakkede kolonner8,9,11,12. For laboratorieskalaisolering forventes membrankromatografi således at reducere isolationstiden med flere timer og øge produktfangst sammenlignet med kolonnekromatografi. Desuden er matematiske modeller for IgG adsorption i membran adsorbere blevet foreslået8,9,11,12, hvilket giver slutbrugerne mulighed for at forudsige ydeevnen i laboratoriet.
En anden flaskehals i grundlæggende videnskab og teknik laboratorier er karakteriseringen af Fc-udtrykke protein. Men, udvælgelse af passende metoder til at fuldføre karakterisering assays, såsom vestlige blots, kan i høj grad reducere den tid, der bruges på produkttest. For eksempel kan halvdry-overførsel i et diskontinuerligt buffersystem udføre elektrofobtisk overførsel af proteiner fra en SDS-PAGE gel til en polyvinyldifluoridin (PVDF) membran i løbet af få minutter i modsætning til 1-2 timers tankoverførsel (våd) overførsel i et kontinuerligt buffersystem13.
En hurtig, billig og effektiv protokol til at isolere og karakterisere et kimært fusionsprotein, der udtrykker et Fc-fragment af menneskelig IgG, er beskrevet heri. Det meste udstyr er let tilgængeligt i grundlæggende biomedicinsk videnskab, biologi, kemi og (bio)kemitekniske laboratorier, og alle reagenser er kommercielt tilgængelige. Selv om repræsentative resultater blev genereret fra isolation og karakterisering af en murine galectin-1/human Fc kimære fusion protein (Gal-1hFc), den strømlinede protokol kan anvendes til isolering af andre molekyler, der besidder en Fc fragment, såsom antistoffer, Fc fragmenter selv, eller andre Fc fusion proteiner. Vores forbedringer reducerede behandlingstiden dramatisk (så få som 4 timer, men mere typisk ~ 9 timer sammenlignet med 20 + arbejdstimer), samtidig med at vi opnåede lignende eller forbedret produktgendannelse sammenlignet med standardmetoder.

<table border="0" cellpadding="0" cellspacing="0" width="822">
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	</colgroup>
	<tbody>
		<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

Laboratorieskala til industriel rensning af biomolekyler fra cellekultur supernatants og lysed celleopløsninger kan opnås ved hjælp af affinitetskromatografi. Mens affinitet kromatografi ved hjælp af porøse protein A agarose perler pakket i kolonner er velsagtens den mest almindelige metode til laboratorieskala isolering af antistoffer og rekombinant proteiner udtrykke Fc fragmenter af IgG, kan det være en tidskrævende og dyr proces. Tid og finansielle begrænsninger er især skræmmende i små grundlæggende videnskab labs, der skal inddrive hundredvis af mikrogram til milligram mængder af protein fra fortyndede løsninger, men mangler adgang til højtryksvæske leveringssystemer og / eller personale med ekspertise i bioseparationer. Desuden kan produktkvantificering og karakterisering også forlænge behandlingstiden for meget over flere arbejdsdage og oppuste udgifterne (forbrugsvarer, lønninger osv.). Derfor er der brug for en hurtig, billig, men effektiv protokol til laboratorieskalaisolering og karakterisering af antistoffer og andre proteiner, der besidder et Fc-fragment. Til dette formål har vi udarbejdet en protokol, der kan udfyldes af teknisk personale med begrænset erfaring på mindre end 9 timer (ca. en arbejdsdag) og så hurtigt som 4 timer, i modsætning til traditionelle metoder, der kræver 20 + arbejdstid. Det meste nødvendige udstyr er let tilgængeligt i standard biomedicinsk videnskab, biokemi og (bio)kemitekniske laboratorier, og alle reagenser er kommercielt tilgængelige. For at demonstrere denne protokol præsenteres repræsentative resultater, hvor kimær murine galectin-1 smeltet til human Fc (Gal-1hFc) fra cellekultur supernatant blev isoleret ved hjælp af et protein A membran adsorber. Renset Gal-1hFc blev kvantificeret ved hjælp af en fremskyndet vestlig blotting analyseprocedure og karakteriseret ved hjælp af flow cytometry. Den strømlinede arbejdsgang kan ændres for andre Fc-udtrykkende proteiner, såsom antistoffer, og / eller ændres til at omfatte alternative kvantificerings- og karakteriseringsmetoder.

Den strømlinede protokol for isolation og karakterisering af Fc-udtrykke proteiner er rutinemæssigt bruges til at behandle kimære murine galectin-1 fusioneret til menneskelige Fc (Gal-1hFc) fra fortyndet cellekultur supernatants. Rutediagrammet i Figur 2 illustrerer arbejdsprocessen og klokkeslættet for hvert trin i protokollen. For et typisk parti på 300 ml supernatant er den samlede behandlingstid ca. 9 timer, når den valgfrie flowcytometritest af elueringsfraktioner udføres. Hvis al flowcytomet...

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

Sammenlignet med traditionel affinitetskromatografi ved hjælp af protein A agarose perlepakkede søjler kan protein A-membrane adsorbere betydeligt fremskynde laboratorieskala isolering af antistoffer og andre FC fragment-udtrykke proteiner. Passende analyse- og kvantificeringsmetoder kan yderligere fremskynde proteinforarbejdningen, så isolation/karakterisering kan gennemføres på én arbejdsdag i stedet for 20+ arbejdstimer.

Isolation og karakterisering af Kimære Human Fc-udtrykke proteiner ved hjælp af Protein A Membrane Adsorbers og en strømlinet Workflow

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

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