Name: A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells (Video) | JoVE Journal
Uploaded: 2015-06-10T15:00:00.000+00:00
Duration: 9 min 16 s
Description: Learn a high-yield method for expressing human granzymes in HEK293T cells using calcium phosphate transfection and chromatography purification. This cost-efficient system produces fully glycosylated, enzymatically active proteases for immune research.

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5 minutes

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 sites (Figure 1). 
	NOTE: Use the following primers indicated in Table 1.</li>
	<li>Confirm correct inserts by sequencing. Expand the expression plasmids in DH5&#945; cells and purify using an endotoxin-free plasmid isolation kit and follow the manufacturer&#8217;s instructions.</li>
	<li>Resuspend the purified plasmids in endotoxin-free, sterile water at a concentration of 2 mg/ml and store at -20 &#176;C until use.</li>
</ol>
2. Expression of Gzms in HEK293T cells
<ol>
	<li>Preparation of reagents
	<ol>
		<li>Prepare standard culture medium. To Dulbecco&#39;s Modified Eagle Medium (DMEM) containing high glucose (25 mM), GlutaMax (4 mM), sodium pyruvate (1 mM) add 10% of standard fetal calf serum (FCS), penicillin (100 units/ml), streptomycin (100 &#956;g/ml).</li>
		<li>Prepare transfection medium. To culture medium without penicillin-streptomycin add 25 &#956;g/ml chloroquine (add freshly on the day of transfection from 1,000x stocks in PBS, stored at -20 &#176;C).</li>
		<li>Prepare serum-free culture medium: To serum-free medium for HEK293 cells add glutamine (4 mM), penicillin (100 units/ml), streptomycin (100 &#956;g/ml), and 2.5mg/L amphotericin B.</li>
		<li>Prepare the solutions described in Table 2 and filter with a 0.45 &#956;m filter before use:</li>
	</ol>
	</li>
	<li>Expanding HEK293T cells
	<ol>
		<li>Grow HEK293T cells in 20 ml culture medium using 150 x 25 mm tissue culture dishes.</li>
		<li>Split cells at 80% confluency (split-ratio of 1:4, usually every 3rd day). Cells loosely attach, they can be mechanically detached without trypsinization by rigorously pipetting up and down.</li>
		<li>Plate cells the night before transfection to give 60-70% confluency at the day of transfection (seeding density ~2 x 105cells/ml). A usual preparation size consists of 20 to 25 culture dishes.</li>
	</ol>
	</li>
	<li>Calcium-phosphate transfection
	<ol>
		<li>1 hr prior to transfection, change the standard culture medium to 20 ml transfection medium. Near the end of this 1 hr incubation step, mix 400 &#956;g of plasmid DNA with 10.95 ml ddH20 and 1.55 ml of 2M CaCl2 in 50 ml tubes (1 tube/5 dishes).</li>
		<li>1 to 2 min prior to the transfection, add 12.5 ml 2x HBS to 12.5 ml of the DNA-Calcium solution, mix by inversion of the tubes and incubate for 30 sec at RT.</li>
		<li>Add the mixture prepared in 2.3.2 directly to the cells (5 ml per dish), drop-wise into the medium. Sprinkle evenly over the entire area, turning the medium to a slightly orange color.</li>
		<li>Incubate the culture dishes for 7 to 11 hr in a tissue culture incubator (37&#176;C, 5% CO2 in humidified air). After the incubation, a fine precipitate is visible. Remove the transfection medium, rinse carefully with pre-warmed PBS and add 20 ml serum-free culture medium prepared in 2.1.3. Incubate for 72 hr in a tissue culture incubator.</li>
		<li>Analyze the transfection and expression efficiency by running a sample (~20 &#956;l) of the cell supernatant on SDS-PAGE and staining with Coomassie Brilliant Blue to visualize a granzyme band.</li>
	</ol>
	</li>
	<li>Purification of Gzms from culture supernatant by Nickel-IMAC
	<ol>
		<li>After the incubation, decant the cell culture supernatants into 250 ml tubes and clear by centrifugation. Carry out a first spin (400 x g, 10 min, 4 &#176;C) that will clear the medium from detached cells. Transfer the supernatant in fresh 250 ml tubes and spin at 4,000 x g, 30 min at 4 &#176;C to remove any remaining cell debris.</li>
		<li>Add 5 ml of 5 M NaCl, 6.25 ml of 2 M Tris-Base (pH 8) and 1 ml of 0.25 M NiSO4per 250 ml of cleared supernatant. Filter the supernatant using a 500 ml Vacuum filter unit (0.45 mm).</li>
		<li>Equilibrate a Nickel-IMAC column with His-binding buffer A (10 column volumes, CV) using a suitable FPLC system.</li>
		<li>Apply the cleared supernatant to the equilibrated column at a flow rate of 0.5 ml/min at 4 &#176;C.</li>
		<li>After the supernatant was applied, wash column with His-binding buffer until UV absorbance baseline is reached (usually 10 CV).</li>
		<li>Elute Gzms with a linear 20 ml-gradient (0 to 250 mM imidazole) at a flow rate of 0.5 ml/min while collecting 2 ml-fractions. Analyze the elution fractions by SDS-PAGE and Coomassie staining.</li>
	</ol>
	</li>
	<li>Enterokinase (EK) treatment and final clean-up by cation exchange chromatography
	<ol>
		<li>Pool Gzm containing fractions in a dialyzing tube with &#8804;10 MWCO. Store a small sample at -20 &#176;C as pre-EK control.</li>
		<li>Add EK at 0.02 unit/50 ml of initial supernatant directly to the pooled fraction in the dialysis tube and dialyze O/N (at least 16 hr) at RT in EK-buffer (4 L, change buffer once).</li>
		<li>The next day, analyze EK treated Gzms in comparison to the pre-EK control by SDS-PAGE and Coomassie staining.</li>
		<li>When N-terminal processing is complete, change dialysis buffer to S buffer A (4 L) and dialyze for another 4 hr at RT. Filter dialysate (0.45 &#956;m).</li>
		<li>Equilibrate a S-column with S buffer A. Load the sample on the column with at a flow rate of 0.5 ml/min at 4 &#176;C.</li>
		<li>After sample loading, wash the column with S buffer A until UV absorbance baseline is reached (about 10 CV). Elute the Gzms with a 30 ml linear gradient (150 to 1,000 mM NaCl). GzmA elutes at ~650 mM NaCl, GzmB at ~700 mM NaCl and GzmM at ~750 mM NaCl.</li>
		<li>Analyze elution fractions by SDS-PAGE and colorimetric assays (see below). Pool fractions containing Gzms and concentrate (about 30-fold, to a concentration of about 100 &#956;M) in spin filters (15 ml, 10 MWCO). Aliquot the concentrated Gzm preparations and store at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
3. Testing Gzm activity
<ol>
	<li>Preparation of reagents
	<ol>
		<li>Prepare colorimetric assay buffer: 50 mM Tris-Base, pH 7.
		<ol>
			<li>For the measurement of GzmA, add N-a-CBZ-L-Lysine thiobenzyl ester (S-Bzl) ( BLT) to the assay buffer to a final concentration of 0.2 mM and 5,5&#8242;-dithio-bis(2-nitrobenzoic acid) (DTNB) to a concentration of 0.22 mM. For GzmB, include 0.2 mM Boc-Ala-Ala-Asp-S-Bzl ( AAD) and 0.22 mM DTNB. For GzmM, include 0.2 mM Suc-Ala-Ala-Pro-Leu-p-nitroanilide (pNA) ( AAPL). Synthetic peptides are added from appropriate stock solutions (in DMSO, stored at -20 &#176;C) to the buffer freshly before the assay.</li>
		</ol>
		</li>
		<li>Prepare cleavage assay buffer: 100 mM NaCl, 50 mM Tris-Base, pH 7.5</li>
	</ol>
	</li>
	<li>Colorimetric assays
	<ol>
		<li>Distribute small Gzm samples (&#60;5 &#956;l) from elution fractions or concentrated stocks in the 96-well flat-bottom plates. Include a positive control (tested Gzm preparations or crude NK cell lysates) and a negative control (buffer only).</li>
		<li>Add 100 &#956;l of assay buffer to each well (using a multi-channel pipette). Incubate for 10 min at 37 &#176;C. Measure OD at 405 nm in a microplate reader.</li>
	</ol>
	</li>
	<li>Cleavage assay
	<ol>
		<li>For cleavage assays using purified substrates, co-incubate a protein substrate (native or recombinant, 100 - 400 nM; a few examples of efficient Gzm substrates are presented in the result section and in the note at the end of this section) with serial dilutions of a Gzm (starting at 400 nM) in assay buffer for various times. Analyze by SDS-PAGE and Coomassie or silver staining; or by western blotting using specific antibodies.</li>
		<li>For cleavage assays using cell lysates, suspend 106 HeLa cells (or any available tumor cell line) in 1 ml of assay buffer and lyse by freezing in an ethanol/dry ice bath and thawing at 37 &#176;C three times. Remove cell debris by centrifugation (15,000 x g for 10 min at 4 &#176;C).</li>
		<li>Co-incubate the cleared lysate with Gzms at various concentrations and times as above. Cleavage is detected by immunoblotting using appropriate antibodies. 
		&#8203;NOTE: A few examples of bona fide substrates for which commercial antibodies are available: Bid (BH3-interacting domain death agonist) and caspase 3 cleaved by GzmB27,28; multiple heterogeneous nuclear ribonucleoproteins (hnRNPA1, A2 and U) cleaved by GzmA29; cytomegalovirus phosphoprotein 71 by GzmM30.</li>
	</ol>
	</li>
</ol>

<ol><li>Chowdhury, D., Lieberman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Death+by+a+thousand+cuts%3A+granzyme+pathways+of+programmed+cell+death%5BTitle%5D)+AND+%22Annu+Rev+Immunol%22%5BJournal%5D)">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/pubmed?term=((Perforin%3A+a+key+pore-forming+protein+for+immune+control+of+viruses+and+cancer%5BTitle%5D)+AND+%22Subcell+Biochem%22%5BJournal%5D)">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/pubmed?term=((Biology+and+clinical+relevance+of+granulysin%5BTitle%5D)+AND+%22Tissue+Antigens%22%5BJournal%5D)">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/pubmed?term=((Orphan+granzymes+find+a+home%5BTitle%5D)+AND+%22Immunol+Rev%22%5BJournal%5D)">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/pubmed?term=((Granzyme+A+activates+another+way+to+die%5BTitle%5D)+AND+%22Immunol+Rev%22%5BJournal%5D)">Granzyme A activates another way to die.</a> Immunol Rev. 235, 93-104 (2010).</li>
<li>Ewen, C. L., Kane, K. P., Bleackley, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+quarter+century+of+granzymes%5BTitle%5D)+AND+%22Cell+death+and+differentiation%22%5BJournal%5D)">A quarter century of granzymes.</a> Cell death and differentiation. 19, 28-35 (2012).</li>
<li>Hiebert, P. R., Granville, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Granzyme+B+in+injury%2C+inflammation%2C+and+repair%5BTitle%5D)+AND+%22Trends+Mol+Med%22%5BJournal%5D)">Granzyme B in injury, inflammation, and repair.</a> Trends Mol Med. 18, 732-741 (2012).</li>
<li>Afonina, I. S., Cullen, S. P., Martin, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytotoxic+and+non-cytotoxic+roles+of+the+CTL%2FNK+protease+granzyme%5BTitle%5D)+AND+%22B.+Immunol+Rev%22%5BJournal%5D)">Cytotoxic and non-cytotoxic roles of the CTL/NK protease granzyme.</a> B. Immunol Rev. 235, 105-116 (2010).</li>
<li>Walch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytotoxic+cells+kill+intracellular+bacteria+through+granulysin-mediated+delivery+of+granzymes%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Cytotoxic cells kill intracellular bacteria through granulysin-mediated delivery of granzymes.</a> Cell. 157, 1309-1323 (2014).</li>
<li>Lee, W. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Invariant+natural+killer+T+cells+act+as+an+extravascular+cytotoxic+barrier+for+joint-invading+Lyme+Borrelia%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Invariant natural killer T cells act as an extravascular cytotoxic barrier for joint-invading Lyme Borrelia.</a> Proc Natl Acad Sci U S A. , (2014).</li>
<li>Kapelski, S., de Almeida, M., Fischer, R., Barth, S., Fendel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antimalarial+activity+of+granzyme+B+and+its+targeted+delivery+by+a+granzyme+B-scFv+fusion+protein%5BTitle%5D)+AND+%22Antimicrob+Agents+Chemother%22%5BJournal%5D)">Antimalarial activity of granzyme B and its targeted delivery by a granzyme B-scFv fusion protein.</a> Antimicrob Agents Chemother. , (2014).</li>
<li>Thiery, J., Walch, M., Jensen, D. K., Martinvalet, D., Lieberman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+cytotoxic+T+cell+and+NK+granules+and+purification+of+their+effector+proteins%5BTitle%5D)+AND+%22Curr+Protoc+Cell+Biol%22%5BJournal%5D)">Isolation of cytotoxic T cell and NK granules and purification of their effector proteins.</a> Curr Protoc Cell Biol. Chapter 3 (Unit3 37), (2010).</li>
<li>Shi, L., Yang, X., Froelich, C. J., Greenberg, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Purification+and+use+of+granzyme+B%5BTitle%5D)+AND+%22Methods+Enzymol%22%5BJournal%5D)">Purification and use of granzyme B.</a> Methods Enzymol. 322, 125-143 (2000).</li>
<li>Masson, D., Tschopp, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+family+of+serine+esterases+in+lytic+granules+of+cytolytic+T+lymphocytes%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">A family of serine esterases in lytic granules of cytolytic T lymphocytes.</a> Cell. 49, 679-685 (1987).</li>
<li>Lorentsen, R. H., Fynbo, C. H., Thogersen, H. C., Etzerodt, M., Holtet, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression%2C+refolding%2C+and+purification+of+recombinant+human+granzyme+B%5BTitle%5D)+AND+%22Protein+Expr+Purif%22%5BJournal%5D)">Expression, refolding, and purification of recombinant human granzyme B.</a> Protein Expr Purif. 39, 18-26 (2005).</li>
<li>Sun, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+and+purification+of+recombinant+human+granzyme+B+from+Pichia+pastoris%5BTitle%5D)+AND+%22Biochem+Biophys+Res+Commun%22%5BJournal%5D)">Expression and purification of recombinant human granzyme B from Pichia pastoris.</a> Biochem Biophys Res Commun. 261, 251-255 (1999).</li>
<li>Xia, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+and+purification+of+enzymatically+active+recombinant+granzyme+B+in+a+baculovirus+system%5BTitle%5D)+AND+%22Biochem+Biophys+Res+Commun%22%5BJournal%5D)">Expression and purification of enzymatically active recombinant granzyme B in a baculovirus system.</a> Biochem Biophys Res Commun. 243, 384-389 (1998).</li>
<li>Stahnke, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Granzyme+B-H22%28scFv%29%2C+a+human+immunotoxin+targeting+CD64+in+acute+myeloid+leukemia+of+monocytic+subtypes%5BTitle%5D)+AND+%22Mol+Cancer+Ther%22%5BJournal%5D)">Granzyme B-H22(scFv), a human immunotoxin targeting CD64 in acute myeloid leukemia of monocytic subtypes.</a> Mol Cancer Ther. 7, 2924-2932 (2008).</li>
<li>Gehrmann, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+expression+and+purification+system+for+the+production+of+enzymatic+and+biologically+active+human+granzyme%5BTitle%5D)+AND+%22B.+J+Immunol+Methods%22%5BJournal%5D)">A novel expression and purification system for the production of enzymatic and biologically active human granzyme.</a> B. J Immunol Methods. 371, 8-17 (2011).</li>
<li>Metkar, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytotoxic+cell+granule-mediated+apoptosis%3A+perforin+delivers+granzyme+B-serglycin+complexes+into+target+cells+without+plasma+membrane+pore+formation%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">Cytotoxic cell granule-mediated apoptosis: perforin delivers granzyme B-serglycin complexes into target cells without plasma membrane pore formation.</a> Immunity. 16, 417-428 (2002).</li>
<li>Motyka, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mannose+6-phosphate%2Finsulin-like+growth+factor+II+receptor+is+a+death+receptor+for+granzyme+B+during+cytotoxic+T+cell-induced+apoptosis%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Mannose 6-phosphate/insulin-like growth factor II receptor is a death receptor for granzyme B during cytotoxic T cell-induced apoptosis.</a> Cell. 103, 491-500 (2000).</li>
<li>Pinkoski, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Entry+and+trafficking+of+granzyme+B+in+target+cells+during+granzyme+B-perforin-mediated+apoptosis%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">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. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+time-+and+cost-efficient+system+for+high-level+protein+production+in+mammalian+cells%5BTitle%5D)+AND+%22Acta+Crystallogr+D+Biol+Crystallogr%22%5BJournal%5D)">A time- and cost-efficient system for high-level protein production in mammalian cells.</a> Acta Crystallogr D Biol Crystallogr. 62, 1243-1250 (2006).</li>
<li>Fukuchi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activity+assays+of+nine+heterogeneous+promoters+in+neural+and+other+cultured+cells%5BTitle%5D)+AND+%22In+Vitro+Cell+Dev+Biol+Anim%22%5BJournal%5D)">Activity assays of nine heterogeneous promoters in neural and other cultured cells.</a> In Vitro Cell Dev Biol Anim. 30 A, 300-305 (1994).</li>
<li>Tran, T. V., Ellis, K. A., Kam, C. M., Hudig, D., Powers, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dipeptidyl+peptidase+I%3A+importance+of+progranzyme+activation+sequences%2C+other+dipeptide+sequences%2C+and+the+N-terminal+amino+group+of+synthetic+substrates+for+enzyme+activity%5BTitle%5D)+AND+%22Arch+Biochem+Biophys%22%5BJournal%5D)">Dipeptidyl peptidase I: importance of progranzyme activation sequences, other dipeptide sequences, and the N-terminal amino group of synthetic substrates for enzyme activity.</a> Arch Biochem Biophys. 403, 160-170 (2002).</li>
<li>Somanchi, S. S., Senyukov, V. V., Denman, C. J., Lee, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expansion%2C+purification%2C+and+functional+assessment+of+human+peripheral+blood+NK+cells%5BTitle%5D)+AND+%22Journal+of+visualized+experiments+%3A+JoVE%22%5BJournal%5D)">Expansion, purification, and functional assessment of human peripheral blood NK cells.</a> Journal of visualized experiments : JoVE. , (2011).</li>
<li>Barry, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Granzyme+B+short-circuits+the+need+for+caspase+8+activity+during+granule-mediated+cytotoxic+T-lymphocyte+killing+by+directly+cleaving+Bid%5BTitle%5D)+AND+%22Mol+Cell+Biol%22%5BJournal%5D)">Granzyme B short-circuits the need for caspase 8 activity during granule-mediated cytotoxic T-lymphocyte killing by directly cleaving Bid.</a> Mol Cell Biol. 20, 3781-3794 (2000).</li>
<li>Darmon, A. J., Nicholson, D. W., Bleackley, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activation+of+the+apoptotic+protease+CPP32+by+cytotoxic+T-cell-derived+granzyme+B.+%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Activation of the apoptotic protease CPP32 by cytotoxic T-cell-derived granzyme B. </a> Nature. 377, 446-448 (1995).</li>
<li>Rajani, D. K., Walch, M., Martinvalet, D., Thomas, M. P., Lieberman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alterations+in+RNA+processing+during+immune-mediated+programmed+cell+death%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Alterations in RNA processing during immune-mediated programmed cell death.</a> Proc Natl Acad Sci U S A. 109, 8688-8693 (2012).</li>
<li>Domselaar, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Noncytotoxic+inhibition+of+cytomegalovirus+replication+through+NK+cell+protease+granzyme+M-mediated+cleavage+of+viral+phosphoprotein+71%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Noncytotoxic inhibition of cytomegalovirus replication through NK cell protease granzyme M-mediated cleavage of viral phosphoprotein 71.</a> J Immunol. 185, 7605-7613 (2010).</li>
<li>Thiery, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perforin+activates+clathrin-+and+dynamin-dependent+endocytosis%2C+which+is+required+for+plasma+membrane+repair+and+delivery+of+granzyme+B+for+granzyme-mediated+apoptosis%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Perforin activates clathrin- and dynamin-dependent endocytosis, which is required for plasma membrane repair and delivery of granzyme B for granzyme-mediated apoptosis.</a> Blood. 115, 1582-1593 (2010).</li>
<li>Thiery, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perforin+pores+in+the+endosomal+membrane+trigger+the+release+of+endocytosed+granzyme+B+into+the+cytosol+of+target+cells%5BTitle%5D)+AND+%22Nat+Immunol%22%5BJournal%5D)">Perforin pores in the endosomal membrane trigger the release of endocytosed granzyme B into the cytosol of target cells.</a> Nat Immunol. 12, 770-777 (2011).</li>
<li>Thomas, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Leukocyte+protease+binding+to+nucleic+acids+promotes+nuclear+localization+and+cleavage+of+nucleic+acid+binding+proteins%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">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/pubmed?term=((Granzyme+B-induced+mitochondrial+ROS+are+required+for+apoptosis%5BTitle%5D)+AND+%22Cell+death+and+differentiation%22%5BJournal%5D)">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. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+of+human+recombinant+granzyme+A+zymogen+and+its+activation+by+the+cysteine+proteinase+cathepsin+C%5BTitle%5D)+AND+%22The+Journal+of+biological+chemistry%22%5BJournal%5D)">Expression of human recombinant granzyme A zymogen and its activation by the cysteine proteinase cathepsin C.</a> The Journal of biological chemistry. 271, 9281-9286 (1996).</li>
<li>Hagn, M., Sutton, V. R., Trapani, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+colorimetric+assay+that+specifically+measures+Granzyme+B+proteolytic+activity%3A+hydrolysis+of+Boc-Ala-Ala-Asp-S-Bzl%5BTitle%5D)+AND+%22Journal+of+visualized+experiments+%3A+JoVE%22%5BJournal%5D)">A colorimetric assay that specifically measures Granzyme B proteolytic activity: hydrolysis of Boc-Ala-Ala-Asp-S-Bzl.</a> Journal of visualized experiments : JoVE. , e52419(2014).</li>
<li>Huang, M. T., Gorman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intervening+sequences+increase+efficiency+of+RNA+3%27+processing+and+accumulation+of+cytoplasmic+RNA%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">Intervening sequences increase efficiency of RNA 3' processing and accumulation of cytoplasmic RNA.</a> Nucleic Acids Res. 18, 937-947 (1990).</li>
<li>Luthman, H., Magnusson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+efficiency+polyoma+DNA+transfection+of+chloroquine+treated+cells%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">High efficiency polyoma DNA transfection of chloroquine treated cells.</a> Nucleic Acids Res. 11, 1295-1308 (1983).</li>
<li>Magee, A. I., Grant, D. A., Hermon-Taylor, J., Offord, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Specific+one-stage+method+for+assay+of+enterokinase+activity+by+release+of+radiolabelled+activation+peptides+from+alpha-N-%5B3H%5Dacetyl-trypsinogen+and+the+effect+of+calcium+ions+on+the+enzyme+activity%5BTitle%5D)+AND+%22Biochem+J%22%5BJournal%5D)">Specific one-stage method for assay of enterokinase activity by release of radiolabelled activation peptides from alpha-N-[3H]acetyl-trypsinogen and the effect of calcium ions on the enzyme activity.</a> Biochem J. 197, 239-244 (1981).</li>
</ol>

The authors have nothing to disclose.

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><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, &amp;ge;20&amp;nbsp;units/mg protein</td></tr><tr><td>HisTrap Excel 5 ml column</td><td>GE Healthcare</td><td>17-3712-06&amp;nbsp;</td><td>Nickel IMAC</td></tr><tr><td>HiTrap SP HP 5 ml column</td><td>GE Healthcare</td><td>17-1152-01&amp;nbsp;</td><td>S column</td></tr><tr><td>N-&amp;alpha;-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&amp;prime;-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

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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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 ...

The overall goal of this procedure is to produce high yields of folic glycosylated and enzymatically active granzymes in human embryonic kidney 2 93 T cells. This is accomplished by first expanding HEC 2 93 T cells in appropriate tissue culture dishes. The usual preparation size after the expansion consists of 20 to 25 culture dishes.The second step is to transiently transfect the expanded cells with a granzyme expression plasmid using the efficient calcium phosphate method. Next, the cell supernat are harvested and the secreted grand enzymes are purified by immobilized metal affinity chromatography. The final steps are to activate the grand enzymes by enter kinase treatment and to further purify the proteases via cation exchange chromatography.Ultimately, enzymatic activity is tested using a rapid and reliable chole metric assay As the availability of active protease in sufficient amounts is a limiting factor in granzyme research. This method can help answer key questions in the field, such as characterization of new cell death pathways in different organisms, or identification of novel protease substrates in whole proteome approaches To express grand enzymes. First grow HEC 2 93 T cells in 20 milliliters of culture medium, using 150 by 25 millimeter tissue culture dishes split the cells at 80%Co fluency cells loosely attach, but can be mechanically detached without trypsin by rigorously pipetting up and down plate.The cells the night before transfection to give 60 to 70%co fluency at the day of transfection. A usual preparation size consists of 20 to 25 culture dishes. One hour prior to transfection changed the standard culture medium to 20 milliliters of transfection medium near the end of the one hour incubation step mixed 400 micrograms of plasma DNA with 10.95 milliliters of double distilled water and 1.55 milliliters of two molar calcium chloride in 50 milliliter tubes one to two minutes prior to the transfection.At 12.5 milliliters of two x heaps buffered saline to 12.5 milliliters of the DNA calcium solution mixed by inversion of the tubes before incubating for 30 seconds. At room temperature, add this mixture, dropwise directly to the cells at five milliliters per dish, sprinkle evenly over the entire area, turning the medium to a slightly orange color. Incubate the culture dishes for seven to 11 hours in a tissue culture incubator.After the incubation, a fine precipitate is visible. Remove the transfection medium. Rinse carefully with prewarm PBS and add 20 milliliters of serum free culture medium before incubating for 72 hours in a tissue culture incubator, analyze the transfection and expression efficiency by running a sample of the cell supernatant on SDS page and staining with kumasi.Brilliant blue. To visualize a granzyme band. After the incubation decant the cell culture supernatants into 250 milliliter tubes and clear by centrifugation.Carry out a first spin that will clear the medium from the detached cells. Transfer the supernatant into fresh 250 milliliter tubes and spin at 4, 000 Gs for 30 minutes at four degrees Celsius to remove any remaining cell debris. Next, add five milliliters of sodium chloride, 6.25 milliliters of tris base and one milliliter of nickel sulfate plus 250 milliliters of cleared supernatant.Filter the supernatant using a 500 milliliter vacuum filter unit. Equilibrate a nickel immobilized metal ion affinity chromatography column with his binding buffer. A using a suitable FPLC system.Then apply the cleared supernatant to the equilibrated column at a flow rate of 0.5 milliliters per minute at four degrees Celsius. After the supernatant is applied, wash the column with his binding buffer until the ultraviolet or UV absorbent baseline is reached. Elute the granzymes with a linear 20 milliliter ILE gradient at a flow rate of 0.5 milliliters per minute while collecting two milliliter fractions.Analyze the elucian fractions by SDS page and kumasi staining. Pull the grand zyme containing fractions in a dazing tube. Store a small sample at minus 20 degrees Celsius as the pre entero kinase control.Add entero kinase at 0.02 units per 50 milliliters of initial supernatant directly to the pooled fraction in the dialysis tube and dialyze overnight at room temperature in entero kinase buffer. The next day analyze kinase treated grand in comparison to the pre enterer kinase control by SDS page and kumasi blue staining. When end terminal processing is complete.Change the dialysis buffer to S buffer A and dialyze for another four hours at room temperature before filtering the DIALATE eight. Next, equilibrate an S column with S buffer. A.Load the sample on the column at a flow rate of 0.5 milliliters per minute at four degrees celsius after sample loading.Wash the column with S buffer A until UV absorbent baseline is reached. Elute the granzymes with a 30 milliliter linear sodium chloride gradient granzyme a elutes at approximately 650 millimolar sodium chloride granzyme B at approximately 700 millimolar sodium chloride and granzyme M at approximately 750 millimolar sodium chloride. Pool the fractions containing granzymes and concentrate to about 100 micromolar in spin filters, aliquot the concentrated granzyme preparations and store at minus 80 degrees Celsius.Next, analyze elucian fractions by SDS page and chole metric assays to perform the chole metric assays distribute small granzyme samples from ellucian fractions or concentrated stocks in 96. Well flat bottom plates include a positive control and a negative control. Add 100 microliters of assay buffer to each.Well incubate the plate for 10 minutes at 37 degrees Celsius. Finally, measure the optical density at 405 nanometers in a microplate reader. This protocol will produce high yields of pure granzymes as demonstrated on this representative kumasi stained SDS page gel showing granzyme BE from the S column.The recombinant granzymes display similar activity as native protease preparations as shown in this color metric assay comparing recombinant and native granzyme B activity. Importantly, endo H treatment indicates that the granzymes produced in HEC 2 93 T cells are fully glycosylated. The recombinant grand enzymes are capable of cleaving macromolecular protein substrates as demonstrated in this representative granzyme a cleavage assay using the bacterial protein substrate and UOCD.Here, the band representing cleaved N-O-U-C-D slowly disappears with increasing granzyme concentration. After watching this video, you should have a good understanding of how to produce fully glycosylated and pro active grand enzymes at high yields in HK 2 93 T cells.

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Research

JoVE Journal

Biology

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

The expression of recombinant proteins by mammalian systems is becoming an attractive method for producing protein complexes for structural biology. Here we present a simple yet highly efficient expression system using suspension grown mammalian cells to purify protein complexes for structural studies.

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

We describe a simple, quantitative colorimetric assay that specifically measures the proteolytic activity of human, mouse or rat Granzyme B (GzmB). This protocol can be easily adapted for determining protease activity of other granule serine proteases by the hydrolysis of other synthetic peptide substrates with an appropriate recognition sequence.

A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl

In this study the expression of a target human recombinant protein in different production platforms was compared. We focused on traditional fermenter-based cultures and on plants, describing the set-up of each system and highlighting, on the basis of the reported results, the inherent limits and advantages for each platform.

A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems

Expression of malarial proteins in cell based systems remains challenging. We demonstrate two step and one step IVT (in vitro translation) cell free expression systems for expressing malarial recombinant rhoptry proteins from HeLa cells. We use a Ni-resin affinity based purification system to purify the rhoptry proteins.

HeLa Based Cell Free Expression Systems for Expression of Plasmodium Rhoptry Proteins

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The purification of ion channels is often challenging, but once achieved, it can potentially allow in vitro investigations of the functions and structures of the channels. Here, we describe the stepwise procedures for the expression and purification of mammalian bestrophin proteins, a family of Ca2+-activated Cl- channels.

Expression and Purification of Mammalian Bestrophin Ion Channels

We provide scalable protocols covering construct design, transient transfection, and expression and purification of full-length human huntingtin protein variants in HEK293 cells.

Efficient and Scalable Production of Full-length Human Huntingtin Variants in Mammalian Cells using a Transient Expression System

His-tag purification, dialysis, and activation are employed to increase yields of soluble, active matrix metalloproteinase-3 catalytic domain protein expression in bacteria. Protein fractions are analyzed via SDS-PAGE gels.

Bacterial Expression and Purification of Human Matrix Metalloproteinase-3 using Affinity Chromatography

Structural and biochemical studies of human membrane transporters require milligram quantities of stable, intact, and homogeneous protein. Here we describe scalable methods to screen, express, and purify human solute carrier transporters using codon-optimized genes.

High-Throughput Expression and Purification of Human Solute Carriers for Structural and Biochemical Studies

Learn a high-yield method for expressing human granzymes in HEK293T cells using calcium phosphate transfection and chromatography purification. This cost-efficient system produces fully glycosylated, enzymatically active proteases for immune research.

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

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