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. Production of the expression plasmid pHLsec-Gzm
<ol>
	<li>Prepare total RNA from human NK cells (primary cells prepared as in26or the NK cell line NK-92 expressing all five human Gzms) using a suitable RNA isolation method and reverse transcribe using a first-strand cDNA synthesize kit, following the manufacturer`s recommendations. Amplify Gzm cDNA using PCR and clone into pHLsec as described in23(kind gift of Radu Aricescu and Yvonne Jones, University of Oxford, UK) using the AgeI and KpnI sit...

<ol>
	<li>Chowdhury, D., Lieberman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Death+by+a+thousand+cuts:+granzyme+pathways+of+programmed+cell+death.">Death by a thousand cuts: granzyme pathways of programmed cell death.</a> Annu Rev Immunol. 26, 389-420 (2008).</li><li>Thiery, J., Lieberman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perforin:+a+key+pore-forming+protein+for+immune+control+of+viruses+and+cancer.">Perforin: a key pore-forming protein for immune control of viruses and cancer.</a> Subcell Biochem. 80, 197-220 (2014).</li><li>Krensky, A. M., Clayberger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+and+clinical+relevance+of+granulysin.">Biology and clinical relevance of granulysin.</a> Tissue Antigens. 73, 193-198 (2009).</li><li>Bovenschen, N., Kummer, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orphan+granzymes+find+a+home.">Orphan granzymes find a home.</a> Immunol Rev. 235, 117-127 (2010).</li><li>Lieberman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granzyme+A+activates+another+way+to+die.">Granzyme A activates another way to die.</a> Immunol Rev. 235, 93-104 (2010).</li><li>Ewen, C. L., Kane, K. P., Bleackley, R. 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Immunol Rev. 235, 105-116 (2010).</li><li>Walch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+cells+kill+intracellular+bacteria+through+granulysin-mediated+delivery+of+granzymes.">Cytotoxic cells kill intracellular bacteria through granulysin-mediated delivery of granzymes.</a> Cell. 157, 1309-1323 (2014).</li><li>Lee, W. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entry+and+trafficking+of+granzyme+B+in+target+cells+during+granzyme+B-perforin-mediated+apoptosis.">Entry and trafficking of granzyme B in target cells during granzyme B-perforin-mediated apoptosis.</a> Blood. 92, 1044-1054 (1998).</li><li>Aricescu, A. R., Lu, W., Jones, E. 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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+protease+binding+to+nucleic+acids+promotes+nuclear+localization+and+cleavage+of+nucleic+acid+binding+proteins.">Leukocyte protease binding to nucleic acids promotes nuclear localization and cleavage of nucleic acid binding proteins.</a> J Immunol. 192, 5390-5397 (2014).</li><li>Jacquemin, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granzyme+B-induced+mitochondrial+ROS+are+required+for+apoptosis.">Granzyme B-induced mitochondrial ROS are required for apoptosis.</a> Cell death and differentiation. , (2014).</li><li>Kummer, J. A., Kamp, A. M., Citarella, F., Horrevoets, A. J., Hack, C. 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The classical and extensively studied role of GzmA and GzmB is the induction of apoptosis in mammalian cells after their cytosolic delivery by the pore-forming protein PFN1. Recently, the cytotoxic spectrum of the Gzms was broadened significantly from mammalian cells to bacteria9,10, as well as to certain parasites11. Furthermore, the non-classical, extracellular functions of GzmA and GzmB as well as the biological significance of the various orphan Gzms are still obscure. Therefore, a ro...

The Gzms are a family of highly homologous serine proteases localized in specialized lysosomes of CTL and NK cells1. The cytotoxic granules of these killer cells also contain the membrane-disrupting proteins PFN and GNLY that are released simultaneously with the Gzms upon recognition of a target cell destined for elimination2,3. There are five Gzms in humans (GzmA, B, H, K and M), and 10 Gzms in mice (GzmA &#8211; G, K, M and N). GzmA and GzmB are the most abundant and extensively studied in humans and mice1. However, more recent studies have begun to investigate the cell-death pathways as well as the additional biological effects mediated by the other, so called orphan Gzms in health and disease4.
The best known function of the Gzms, in particular of GzmA and GzmB, is the induction of programmed cell death in mammalian cells when delivered into the target cells by PFN5,6. However, more recent studies also demonstrated extracellular effects of the Gzms with profound impact on immune regulation and inflammation, independently of cytosolic delivery by PFN7,8. The spectrum of cells that are killed efficiently after cytosolic entry of the Gzms was also recently widened from mammalian cells to bacteria9,10and even certain parasites11. These recent discoveries opened up a whole new field for Gzm researchers. Therefore, a cost-efficient, high-yield mammalian expression system will significantly ease the way for those future studies.
Native human, mouse and rat Gzms have been successfully purified from the granule fraction of CTL and NK cells lines12-14. However, in our hands the yield of such purification techniques is in the range of less than 0.1 mg/L cell culture (unpublished observation and12). Furthermore, the chromatographic resolution of a single Gzm without contamination by other Gzms and/or proteins that are also present in the granules is challenging (unpublished data and12,14). Recombinant Gzms were produced in bacteria15, yeast16, insect cells17and in even in mammalian cells such as HEK 29318,19. Only the mammalian expression systems bear the potential to produce recombinant enzymes with posttranslational modifications identical to the native cytotoxic protein. Posttranslational modifications have been implicated with the specific uptake by endocytosis and the intracellular localization of the protease within target cells20-22. Therefore, by using pHLsec23(a kind gift of Radu Aricescu and Yvonne Jones, University of Oxford, UK) as the plasmid backbone for Gzm expression, we established a simple, time- and cost-efficient system for high-yield protein production in HEK293T cells. pHLsec combines a CMV enhancer with a chicken &#914;-actin promoter; together, these elements demonstrated the strongest promoter activity in various cell lines24. In addition, the plasmid contains a rabbit &#914;-globin intron, optimized Kozak and secretion signals, a Lys-6xHis-tag and a poly-A signal. Inserts can be cloned conveniently between the secretion signal and the Lys-6xHis-tag (Figure 1) ensuring optimal expression and secretion efficiency for proteins lacking appropriate N-terminal domains. For the expression of the Gzms, we replaced the endogenous secretion signal sequence with the secretion signal provided by the vector followed by an enterokinase (EK) site (DDDDK) so that EK treatment activated the secreted Gzms (active Gzms start with the N-terminal amino acid sequence IIGG25). Additionally in favor for this method, HEK293T cells grow rapidly in low priced medium, such as Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM), and are well suited for cost-efficient calcium-phosphate transfection method.

<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.

In the following section we will present a complete documentation of a GzmA preparation to illuminate the method. We also successfully purified GzmB and GzmM, producing similar results in regard to purification efficiency and activity. However, from those later preparations we will only show a few selected pieces of data. GzmB preparations from 293T cells following the current protocol were used in several published studies, highlighting their activity in various biological assay systems9,29,31-34.
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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.

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

a-high-yield-cost-efficient-expression-system-human-granzymes

Research

JoVE Journal

Biology

9.6K Aufrufe.  Boston Children’s Hospital and Harvard Medical School. We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease. 