Name: a high yield and cost-efficient expression system of human granzymes in mammalian cells
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Description: Watch this Scientific Journal Video about A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells at JoVE.com

This work was supported by grants from the Novartis Foundation for Medical-Biological Research and from the Research Pool of the University of Fribourg (to MW). We thank Li Zhao, Zhan Xu, and Solange Kharoubi Hess for technical support, as well as Radu Aricescu and Yvonne Jones (Oxford University, UK) for providing the pHLsec plasmid, and Thomas Sch&#252;rpf (Harvard Medical School) for helpful discussions.

1. Herstellung des Expressionsplasmids pHLsec-GZM <ol><li> Bereiten Gesamt-RNA aus menschlichen NK-Zellen (Primärzellen, wie in 26 oder der NK-Zelllinie NK-92 exprimieren, alle fünf menschlichen Gzms hergestellt) mit einer geeigneten RNA-Isolierungsverfahren und Umkehr transcribe unter Verwendung eines ersten cDNA synthetisieren Satz, nach dem manufacturer` s Empfehlungen. Verstärken GZM cDNA mittels PCR und Klon in pHLsec wie in 23 (Art Geschenk der Radu Aricescu und Yvonne Jones, University ...

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Die klassische und ausgiebig untersucht Rolle GZMA und GzmB ist die Induktion von Apoptose in Säugetierzellen nach ihrer zytosolischen Lieferung durch den porenbildendes Protein PFN 1. Kürzlich wurde die zytotoxische Spektrum der Gzms signifikant von Säugetierzellen, Bakterien 9,10 sowie bestimmte Parasiten 11 verbreitert. Darüber hinaus sind die nicht-klassischen, extrazelluläre Funktionen GZMA und GzmB sowie die biologische Bedeutung der verschiedenen Waisen Gzms noch unklar. Dahe...

Die Gzms sind eine Familie von hoch homologen Serinproteasen in spezialisierten Lysosomen von CTL und NK-Zellen 1 lokalisiert. Die zytotoxischen Granula dieser Killerzellen enthalten auch die Membran-störenden Proteinen PFN und GNLY, die gleichzeitig mit den Gzms bei Erkennung einer Zielzelle für die Eliminierung 2,3 bestimmt werden freigegeben. Es gibt fünf Gzms beim Menschen (GZMA, B, H, K und M) und 10 Gzms in Mäusen (GZMA - G, K, M und N). GZMA und GzmB sind die häufigsten und umfassend in Menschen und Mäusen 1 untersucht. Allerdings haben neuere Studien begonnen, die Zelltod Wege sowie die zusätzlichen biologischen Wirkungen von den anderen, so genannten Orphan-Gzms in Gesundheit und Krankheit 4 vermittelt zu untersuchen. Das am besten bekannte Funktion der Gzms, insbesondere GZMA und GzmB, ist die Induktion von programmiertem Zelltod in Säugerzellen, wenn sie von PFN 5,6 in die Zielzellen geliefert. JedochZeigten neuere Studien auch extrazelluläre Effekte der Gzms mit tiefgreifenden Auswirkungen auf die Immunregulation und Entzündungen, unabhängig von zytosolischen Lieferung durch PFN 7,8. Das Spektrum von Zellen, die effizient nach cytosolischen Eingabe der Gzms getötet wurde vor kurzem auch aus Säugerzellen, Bakterien 9,10 und sogar bestimmte Parasiten 11 verbreitert. Diese jüngsten Entdeckungen auf ein ganz neues Feld für GZM Forscher eröffnet. Daher wird eine kostengünstige, High-Yield-Säugetier-Expressionssystem deutlich den Weg für die künftige Studien erleichtern. Nativen menschlichen, Maus und Ratte Gzms wurden erfolgreich aus der Kornfraktion von CTL und NK-Zellen Linien 12-14 gereinigt. Jedoch in unseren Händen die Ausbeute solcher Reinigungsverfahren ist in dem Bereich von weniger als 0,1 mg / l Zellkultur (unveröffentlichte Beobachtung und 12). Darüber hinaus die chromatographische Auflösung eines einzelnen GZM ohne Verunreinigung durch other Gzms und / oder Proteine, die ebenfalls vorhanden sind, in den Granulaten ist eine Herausforderung (unveröffentlichte Daten und 12,14). Rekombinante Gzms wurden Bakterien 15, Hefe 16, Insektenzellen 17 und auch in Säugerzellen, wie HEK 293 18,19 hergestellt. Nur die Säugetier-Expressionssysteme tragen das Potenzial, rekombinanten Enzyme mit posttranslationalen Modifikationen identisch mit dem nativen zytotoxischen Protein zu produzieren. Posttranslationale Modifikationen der spezifischen Aufnahme durch Endozytose und die intrazelluläre Lokalisation der Protease in Zielzellen 20-22 gebracht. Daher kann unter Verwendung pHLsec 23 (ein freundliches Geschenk von Radu Aricescu und Yvonne Jones, University of Oxford, UK) als Plasmidrückgrat für GZM Ausdruck haben wir ein einfaches, zeit- und kosteneffizientes System für High-Yield-Proteinproduktion in HEK293T-Zellen. pHLsec kombiniert eine CMV-Enhancer mit einem Huhn Β-Actin-Promotor; Zusammen bilden diese Elemente Demonstrationented der stärkste Promotor-Aktivität in verschiedenen Zelllinien 24. Zusätzlich enthält das Plasmid ein Kaninchen Β-Globin-Intron, optimierte Kozak und Sekretionssignalen, einen Lys-6xHis-Tag und einem Poly-A-Signal. Inserts können bequem zwischen dem Sekretionssignal und dem Lys-6xHis-tag (Abbildung 1) eine optimale Expression und Sekretion Effizienz für Proteine ​​fehlen geeignete N-terminalen Domänen geklont werden. Für die Expression der Gzms, die endogene Sekretionssignal-Sequenz mit dem Sekretionssignal von dem Vektor, gefolgt von einer Enterokinase (EK) Website (DDDDK) versehen ist, so dass wir ersetzt EK Behandlung aktiviert die sezernierten Gzms (aktiv Gzms beginnen mit dem N-terminalen Aminosäuresequenz IIGG 25). Zusätzlich zugunsten für dieses Verfahren, HEK293T Zellen schnell wachsen in günstiges Medium, wie Dulbeccos modifiziertes Eagle-Medium (DMEM) und sind für eine kosteneffiziente Calciumphosphat-Transfektionsverfahren gut geeignet.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Invitrogen</td>
			<td>15596-026</td>
			<td>Total RNA isolation kit</td>
		</tr>
		<tr>
			<td>SuperScript II Reverse Transcriptase</td>
			<td>Invitrogen</td>
			<td>18064-014</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA Polymerase</td>
			<td>NEB</td>
			<td>M0530L</td>
			<td></td>
		</tr>
		<tr>
			<td>EndoFree Plasmid Maxi Kit</td>
			<td>QIAGEN</td>
			<td>12362</td>
			<td></td>
		</tr>
		<tr>
			<td>EX-CELL 293 Serum-Free Medium for HEK 293 Cells</td>
			<td>Sigma</td>
			<td>14571C</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, GlutaMAX Supplement, pyruvate</td>
			<td>Gibco</td>
			<td>31966-021</td>
			<td></td>
		</tr>
		<tr>
			<td>SnakeSkin Dialysis Tubing, 10K MWCO</td>
			<td>Thermo Scientific</td>
			<td>68100</td>
			<td></td>
		</tr>
		<tr>
			<td>Enterokinase from bovine intestine</td>
			<td>Sigma</td>
			<td>E4906</td>
			<td>recombinant, &#8805;20&#160;units/mg protein</td>
		</tr>
		<tr>
			<td>HisTrap Excel 5ml-column</td>
			<td>GE Healthcare</td>
			<td>17-3712-06&#160;</td>
			<td>Nickel IMAC</td>
		</tr>
		<tr>
			<td>HiTrap SP HP 5 ml-column</td>
			<td>GE Healthcare</td>
			<td>17-1152-01&#160;</td>
			<td>S column</td>
		</tr>
		<tr>
			<td>N-&#945;-Cbz-L-lysine thiobenzylester (BLT)</td>
			<td>Sigma</td>
			<td>C3647</td>
			<td>GzmA substrate</td>
		</tr>
		<tr>
			<td>Boc-Ala-Ala-Asp-S-Bzl (AAD)</td>
			<td>MP Biomedicals</td>
			<td>2193608 _10mg</td>
			<td>GzmB substrate</td>
		</tr>
		<tr>
			<td>Suc-Ala-Ala-Pro-Leu-p-nitroanilide (AAPL)</td>
			<td>Bachem</td>
			<td></td>
			<td>GzmM substrate</td>
		</tr>
		<tr>
			<td>5,5&#8242;-dithio-bis(2-nitrobenzoic acid) (DTNB)</td>
			<td>Sigma</td>
			<td>D8130</td>
			<td>Ellman`s reagent</td>
		</tr>
	</tbody>
</table>

a high yield and cost-efficient expression system of human granzymes in mammalian cells

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their cytotoxic granule content to eliminate the target cells and the intracellular pathogen. Death of the host cells and intracellular pathogens is triggered by the granule serine proteases, granzymes (Gzms), delivered into the host cell cytosol by the pore forming protein perforin (PFN) and into bacterial pathogens by the prokaryotic membrane disrupting protein granulysin (GNLY). To investigate the molecular mechanisms of target cell death mediated by the Gzms in experimental in-vitro settings, protein expression and purification systems that produce high amounts of active enzymes are necessary. Mammalian secreted protein expression systems imply the potential to produce correctly folded, fully functional protein that bears posttranslational modification, such as glycosylation. Therefore, we used a cost-efficient calcium precipitation method for transient transfection of HEK293T cells with human Gzms cloned into the expression plasmid pHLsec. Gzm purification from the culture supernatant was achieved by immobilized nickel affinity chromatography using the C-terminal polyhistidine tag provided by the vector. The insertion of an enterokinase site at the N-terminus of the protein allowed the generation of active protease that was finally purified by cation exchange chromatography. The system was tested by producing high levels of cytotoxic human Gzm A, B and M and should be capable to produce virtually every enzyme in the human body in high yields.

Im folgenden Abschnitt werden wir eine vollständige Dokumentation eines GZMA Vorbereitung zu präsentieren, um die Methode zu beleuchten. Wir haben auch erfolgreich gereinigt GzmB und GzmM, die ähnliche Ergebnisse in Bezug auf Reinigungsleistung und Aktivität. Doch von diesen später Vorbereitungen werden wir nur zeigen einige ausgewählte Stücke von Daten. Wurden GzmB Präparate aus 293T-Zellen nach dem aktuellen Protokoll in mehreren veröffentlichten Studien verwendet, Hervorhebung ihrer Tätigkeit in verschieden...

Watch this Scientific Journal Video about A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells at JoVE.com

A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells

We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease.

Eine hohe Ausbeute und kosteneffiziente Expression System der Menschen Granzyme in Säugerzellen

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their ...

Research

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Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

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