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>

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<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 robust time- and cost-efficient expression and purification system of the Gzms, as provided by this protocol, will be of great help for these future studies.
The strength of this method is particularly based on the expression vector, pHLsec23, that is used in this system. This plasmid has a high copy number in E. coli, thus enabling efficient DNA production. Its enhancer/promoter elements provide the strongest activity in various cell lines24. It contains an intron within the transcription unit. Introns have been found to enhance gene expression in mammalian cells up to 100-fold37. In addition, the plasmid provides a Kozak consensus and an optimized secretion signal, a Lys-6xHis-tag and a poly-A signal (Figure 1). Inserts can be cloned by using the AgeI and KpnI sites between the secretion signal and the Lys-6xHis tag. Using AgeI and XhoI will avoid the polyHis tag. For the Gzms, it was necessary to insert an enterokinase site at the N-terminus of the proteases enabling activation of the proteases after expression and IMAC purification. For the expression of other enzymes this might not be necessary. The expression plasmid was successfully tested for multiple constructs of various lengths, glycosylation states, and numbers of disulfide bonds, as well as for proteins that contained multiple domains with different folds23.
HEK293T cells grow rapidly in low cost medium and are very well suited for cost-efficient calcium-phosphate transfection. For this protocol, we recommend not to over-passage the HEK293T cells. Frozen stocks of low-passage-cells should be prepared. Expansion takeoff the cells usually took about 8 to 9 days (3 splits) to obtain the desired amount of cells. The cell density at the day of transfection (60 to 70% confluency) was critical for efficient transfection and consequently expression. Also, avoiding antibiotics but adding chloroquine (that inhibits lysosomal DNA degradation) to the medium significantly enhanced efficiency38. We found that transfection efficiency was linearly dependent of the DNA amount saturating only at about 80 &#956;g for a 15-cm dish (unpublished observation). Therefore, we recommend using up to 80 &#956; g of plasmid DNA/dish in this protocol for maximal efficiency. This will amount to about 2 mg of DNA per preparation (corresponding to 2 MaxiPrep columns). The transfection efficiency was most easily monitored by running a sample of culture supernatant (about 20 &#956; l) on SDS-PAGE after the 72 hr incubation period. A protein band of the secreted protease should be visible by Coomassie staining. If the Gzm band was weak and the cells did not detach and appeared viable, the cultures were incubated for another 24 hr before IMAC purification.
Another most critical step was the enterokinase (EK) treatment that activates the enzyme. EK activity is calcium dependent and is inhibited by high concentrations of NaCl39or imidazole (unpublished observation). Therefore, it was crucial to dialyze the eluate from the IMAC against a sufficient volume (20 ml eluate per 2 L of dialysis buffer) of EK buffer containing enough calcium and not more than 50 mM of NaCl at neutral pH. EK treatment is recommended for 16 hr at RT and should be monitored by SDS-PAGE that revealed a mobility shift of the treated protease if the cleavage was complete. Fresh EK was added and the incubation in fresh EK Dialysis buffer was continued if the shift was incomplete. Only if the cleavage was complete, dialysis against S buffer A was started and purification continued. The catalytic light chain of EK has a pI value of 5.2, allowing the complete separation of EK from the Gzms in the final cation exchange chromatography.

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

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.
A typical GzmA purification is shown in Figure 2A. Efficient transfection and expression after 72 hr&#160;was indicated by a detectable Gzm band on a Coomassie-stained SDS-PAGE gel without further concentration of the culture supernatant (~37 kDa for GzmB and GzmM under reducing conditions; ~60/34 kDa for GzmA under non-reducing/reducing conditions). After the IMAC purification the samples were treated with enterokinase (EK). A visible shift of the EK treated sample (post-EK) compared to the pre-EK control was observed. The final purification on the S-column resulted in highly pure Gzm preparations (GzmA in Figure 2A; GzmB and GzmM in Figure 2B) with a single band appearance on Coomassie-stained protein gels. The pooled fractions were concentrated to a final concentration of ~100 &#956;M. Typically, we obtained yields of about 0.5 to 1 mg Gzms per 100 ml culture supernatant. To demonstrate efficient glycosylation, a few initial preparations were treated with Endoglycosidase (Endo) H, which removes high mannose oligosaccharides from N-linked glycoproteins. Native GzmA has a N-linked glycosylation site at Asn-142, to which a high mannose oligosaccharide is bound35. EndoH treatment induced an obvious mobility shift in the Gzm A, as well as GzmB, preparations from HEK293T cells but not in bacterially generated GzmA as analyzed by SDS-PAGE and Coomassie staining (Figure 2C).
Routinely, every Gzm batch was tested for enzymatic activity in colorimetric assays. This rapid and reliable test demonstrates proteolytic activity of the Gzms by the cleavage of small synthetic peptides that is indicated spectrophotometrically by a color change of the assay buffer. Due to different preferences concerning the amino acid residues (P1 position) after which these proteases cleave, the choice of a specific peptide varies among the Gzms. GzmA has trypsin-like activity, cleaving after the basic residues arginine (Arg, R) and lysine (Lys, K). GzmB cleaves preferentially after aspartic acid residues (Asp, D), and GzmM cleaves after leucine (Leu, L) and methionine (Met, M)1. Our preferential peptide choice is BLT-S-Bzl to measure GzmA activity, AAD-S-Bzl for GzmB and AAPL-pNA for GzmM.
The major difference between the thiobenzylester substrates (AAD and BLT) and the p-nitroanilide substrate (AAPL) is the specific chemistry, in that Gzm mediates cleavage of thiobenzylester substrates releases benzyl-mercaptan, which only triggers a chromogenic reaction in its downstream reaction with the chromophore DTNB. Therefore, in the reaction buffers of thiobenzylester substrates DTNB has to be present, while the Gzm-mediated release of p-nitroanilide is sufficient for the colorimetric detection. The detailed method of the colorimetric assays was recently published36. Figure 3A shows the representative BLT and AAPL esterase activity in the elution fractions from the S-column of a GzmA and GzmM preparation, respectively. Colorimetric assay was also used to compare the activity of recombinant to native GzmB preparations. As shown in Figure 3B, recombinant 293T GzmB cleaved the chromogenic substrate with similar efficiency compared to native GzmB purified from a human NK cell line, as recently described12.
To more specifically test Gzm activity, Gzm cleavage assays using known protein substrates are indicated. These cleavage assays can be performed with purified recombinant or native protein (if available), with cell lysates or most physiological with intact cells. If a protein substrate is available, proteolytic activity can be analyzed in simple co-incubation experiments with the Gzm preparations as this is demonstrated with the known bacterial GzmA and GzmB substrates nuoCD and nuoF9, as well as with the novel human mitochondrial GzmM substrate NDUFAF3 (Figure 4A).
Cell lysates represent another obvious source of multiple Gzm substrates. Commercial antibodies are available against many of the substrates. A fast and simple method to generate cell lysates is by applying multiple freeze/thaw cycles as previously demonstrated33. The use of detergents is not recommended as they can interfere with Gzm activity. In Figure 4B, GzmA mediated cleavage of hnRNPA1 in a HeLa cell lysate is demonstrated in an immunoblot using an anti-hnRNPA1 antibody, as well as an anti-&#914;-actin antibody as loading control.
For cleavage assays (or cytotoxicity assays) in intact cells, the availability of an additional delivery molecule is necessary (PFN or Streptolysin O, SLO, for mammalian cells; GNLY or other antimicrobial peptides for prokaryotic cells). Cleavage assays in intact cells are most challenging as the concentration of the delivery molecules is critical and needs extensive fine tuning. Introducing these complex protocols of apoptosis assays is beyond the scope of this paper. Here, we may only refer to the vast body of literature, as examples12,13.
<img alt="Table 1" src="/files/ftp_upload/52911/52911table1.jpg" /> 
Table 1: Gzm Cloning Primer Sequences.
The primer sequences that were used to clone GzmA, GzmB and GzmM are indicated. Forward primers were designed to introduce an EK site before the N-terminus of the active protease (see Figure 1)
<img alt="Table 2" src="/files/ftp_upload/52911/52911table2.jpg" /> 
Table 2: Stock Solutions and Buffer Compositions.
The recipes of the most important stock solutions and buffers are indicated.
<img alt="Figure 1" src="/files/ftp_upload/52911/52911fig1.jpg" /> 
Figure 1. pHLsec Sequence Critical for Construct Cloning.
In the sequence, a few elements in the vector are highlighted that are important for cloning, secretion, IMAC purification and activation of the proteases: The Kozak consensus and secretion signal sequences, the AgeI and KpnI restriction sites, as well as the C-terminal Lys_6xHis tag. We exemplified GzmA as the insert that is N-terminally fused to an enterokinase (EK) site. For the full map of the complete vector backbone, we refer to the supplementary information in23.
<img alt="Figure 2" src="/files/ftp_upload/52911/52911fig2.jpg" /> 
Figure 2. Expression, Purification and Activation of Fully Glycosylated Human Gzms from HEK293T Cells.
A) shows a series of Coomassie-stained, non-reducing SDS-PAGE gels demonstrating the whole GzmA production process from its secretion in the supernatant to first Nickel-IMAC purification, EK treatment and final polishing via cation exchange chromatography. The culture supernatant was run on SDS-PAGE under reducing (red) and non-reducing (non-red) conditions. GzmA is a homodimer (ssGzmA) that is stabilized by a disulfide bond. The GzmA homodimer (~60 kDa) runs close to contaminating BSA (~66 kDa). Under reducing conditions the GzmA monomer (shGzmA) appears as a ~34 kDa band. To demonstrate the purity of the final GzmB and GzmM preparations, representative Coomassie-stained gels under non reducing conditions are shown in B. Bacterially (E. coli) generated GzmA as well as GzmA and GzmB produced in HEK293T cells were treated with EndoH and analyzed by non-reducing SDS-PAGE and Coomassie staining. Glycosylation of the Gzms produced in HEK293T cells is demonstrated by a mobility shift after EndoH treatment as compared to non-glycosylated GzmA produced in bacteria (C).
<img alt="Figure 3" src="/files/ftp_upload/52911/52911fig3.jpg" /> 
Figure 3. Recombinant Gzms Hydrolyze Synthetic Peptides.
A), small samples of the elution fractions from the final S column were tested for GzmA and GzmM activity in chromogenic assays using BLT and AAPL, respectively, as substrates. Graphs show peptide cleavage that was indicated as an OD increase at 405 nm. The activity peak fractions correlated with the protein peak in the elution as indicated in UV absorbance and SDS-PAGE analysis (Figure 2A and B). B), recombinant 293T and native GzmB (prepared as described12) at indicated concentrations were incubated in presence of the chromogenic substrate AAD. GzmB activity was indicated as an OD change at 405 nm. Graph shows the mean &#177; SEM of our most recent Gzm preparations that were tested in triplicates.
<img alt="Figure 4" src="/files/ftp_upload/52911/52911fig4.jpg" /> 
Figure 4.&#160;Recombinant Gzms Cleave Protein Substrates.
A) Recombinant GST-tagged, bacterial respiratory chain proteins nuoCD and nuoF, as well as the mammalian respiratory chain protein NDUFAF3, were treated with indicated concentration of indicated Gzms for 15 min at 37 &#176;C. 1 &#956;g of purified proteins in 20 &#956;l&#160;reactions were used. Cleavage was analyzed by SDS-PAGE and Coomassie staining. B) HeLa cell lysate (at a protein concentration of ~1 mg/ml) was incubated with indicated concentrations of recombinant GzmA for 30 min and analyzed by immunoblotting using anti hnRNPA1 and &#914;-actin antibodies.

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

Nanyang Technological University

Research

JoVE Journal

Biology

9.8K Views.  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. 

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

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

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is the most studied granzyme in humans and mice and therefore, researchers need specific and reliable tools to study its function and role in pathophysiology. This especially necessitates assays that do not recognize proteases such as caspases or other granzymes that are structurally or functionally related. Here, we apply GzmB&#8217;s preference for cleavage after aspartic acid residues in a colorimetric assay using the peptide thioester Boc-Ala-Ala-Asp-S-Bzl. GzmB is the only mammalian serine protease capable of cleaving this substrate. The substrate is cleaved with similar efficiency by human, mouse and rat GzmB, a property not shared by other commercially available peptide substrates, even some that are advertised as being suitable for this purpose. This protocol is demonstrated using unfractionated lysates from activated NK cells or CTL and is also suitable for recombinant proteases generated in a variety of prokaryotic and eukaryotic systems, provided the correct controls are used. This assay is a highly specific method to ascertain the potential pro-apoptotic activity of cytotoxic molecules in mammalian lymphocytes, and of their recombinant counterparts expressed by a variety of methodologies.

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.

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is ...

Chemistry

Expression and Purification of Mammalian Bestrophin Ion Channels

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- channel (CaCC) predominantly expressed in retinal pigment epithelium (RPE). The physiological and pathological significance of BEST1 is highlighted by the fact that over 200 distinct mutations in the BEST1 gene have been genetically linked to a spectrum of at least five retinal degenerative disorders, such as Best vitelliform macular dystrophy (Best disease). Therefore, understanding the biophysics of bestrophin channels at the single-molecule level holds tremendous significance. However, obtaining purified mammalian ion channels is often a challenging task. Here, we report a protocol for the expression of mammalian bestrophin proteins with the BacMam baculovirus gene transfer system and their purification by affinity and size-exclusion chromatography. The purified proteins have the potential to be utilized in subsequent functional and structural analyses, such as electrophysiological recording in lipid bilayers and crystallography. Importantly, this pipeline can be adapted to study the functions and structures of other ion channels.

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.

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

Biochemistry

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

Full-length huntingtin (FL HTT) is a large (aa 1-3,144), ubiquitously expressed, polyglutamine (polyQ)-containing protein with a mass of approximately 350 kDa. While the cellular function of FL HTT is not entirely understood, a mutant expansion of the polyQ tract above ~36 repeats is associated with Huntington's disease (HD), with the polyQ length correlating roughly with the age of onset. To better understand the effect of structure on the function of mutant HTT (mHTT), large quantities of the protein are required. Submilligram production of FL HTT in mammalian cells was achieved using doxycycline-inducible stable cell line expression. However, protein production from stable cell lines has limitations that can be overcome with transient transfection methods. 
This paper presents a robust method for low-milligram quantity production of FL HTT and its variants from codon-optimized plasmids by transient transfection using polyethylenimine (PEI). The method is scalable (&gt;10 mg) and consistently yields 1-2 mg/L of cell culture of highly purified FL HTT. Consistent with previous reports, the purified solution state of FL HTT was found to be highly dynamic; the protein has a propensity to form dimers and high-order oligomers. A key to slowing oligomer formation is working quickly to isolate the monomeric fractions from the dimeric and high-order oligomeric fractions during size exclusion chromatography. 
Size exclusion chromatography with multiangle light scattering (SEC-MALS) was used to analyze the dimer and higher-order oligomeric content of purified HTT. No correlation was observed between FL HTT polyQ length (Q23, Q48, and Q73) and oligomer content. The exon1-deleted construct (aa 91-3,144) showed comparable oligomerization propensity to FL HTT (aa 1-3,144). Production, purification, and characterization methods by SEC/MALS-refractive index (RI), sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot, Native PAGE, and Blue Native PAGE are described herein.

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

Full-length huntingtin (FL HTT) is a large (aa 1-3,144), ubiquitously expressed, polyglutamine (polyQ)-containing protein with a mass of approximately 350 ...

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

Matrix metalloproteinases (MMPs) belong to the family of metzincin proteases with central roles in extracellular matrix (ECM) degradation and remodeling, as well as interactions with several growth factors and cytokines. Overexpression of specific MMPs is responsible in several diseases such as cancer, neurodegenerative diseases, and cardiovascular disease. MMPs have been the center of attention recently as targets to develop therapeutics that can treat diseases correlated to MMP overexpression.
To study the MMP mechanism in solution, more facile and robust recombinant protein expression and purification methods are needed for the production of active, soluble MMPs. However, the catalytic domain of most MMPs cannot be expressed in Escherichia coli (E. coli) in soluble form due to lack of posttranslational machinery, whereas mammalian expression systems are usually costly and have lower yields. MMP inclusion bodies must undergo the tedious and laborious process of extensive purification and refolding, significantly reducing the yield of MMPs in native conformation. This paper presents a protocol using Rosetta2(DE3)pLysS (hereafter referred to as R2DP) cells to produce matrix metalloproteinase-3 catalytic domain (MMP-3cd), which contains an N-terminal His-tag followed by pro-domain (Hisx6-pro-MMP-3cd) for use in affinity purification. R2DP cells enhance the expression of eukaryotic proteins through a chloramphenicol-resistant plasmid containing codons normally rare in bacterial expression systems. Compared to the traditional cell line of choice for recombinant protein expression, BL21(DE3), purification using this new strain improved the yield of purified Hisx6-pro-MMP-3cd. Upon activation and desalting, the pro domain is cleaved along with the N-terminal His-tag, providing active MMP-3cd for immediate use in countless in vitro applications. This method does not require expensive equipment or complex fusion proteins and describes rapid production of recombinant human MMPs in bacteria.

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.

Matrix metalloproteinases (MMPs) belong to the family of metzincin proteases with central roles in extracellular matrix (ECM) degradation and remodeling, ...

Strategies for Expressing and Purifying Solute Carrier Transporters

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

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, metabolites, neurotransmitters, and pharmaceuticals. Despite having emerged as attractive therapeutic targets and markers of disease, this group of proteins is still relatively underdrugged by current pharmaceuticals. Drug discovery projects for these transporters are impeded by limited structural, functional, and physiological knowledge, ultimately due to the difficulties in the expression and purification of this class of membrane-embedded proteins. Here, we demonstrate methods to obtain high-purity, milligram quantities of human SLC transporter proteins using&#160;codon-optimized gene sequences. In conjunction with a systematic exploration of construct design and high-throughput expression, these protocols ensure the preservation of the structural integrity and biochemical activity of the target proteins. We also highlight critical steps in the eukaryotic cell expression, affinity purification, and size-exclusion chromatography of these proteins. Ultimately, this workflow yields pure, functionally active, and stable protein preparations suitable for high-resolution structure determination, transport studies, small-molecule engagement assays, and high-throughput in vitro screening.

Author Spotlight: Expression and Purification of Human Solute Carrier Transporters Using Codon-Optimized Genes 

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.

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, ...

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

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

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.

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

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

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F 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.

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two ...

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

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the flexible, low-cost production of high-quality, bioactive products.
In this study, we compared the expression of a target human recombinant protein in traditional fermenter-based cell cultures (bacterial and insect) with plant-based expression systems, both transient and stable.
For each platform, we described the set-up, optimization and length of the production process, the final product quality and the yields and we evaluated provisional production costs, specific for the selected target recombinant protein.
Overall, our results indicate that bacteria are unsuitable for the production of the target protein due to its accumulation within insoluble inclusion bodies. On the other hand, plant-based systems are versatile platforms that allow the production of the selected protein at lower-costs than Baculovirus/insect cell system. In particular, stable transgenic lines displayed the highest-yield of the final product and transient expressing plants the fastest process development. However, not all recombinant proteins may benefit from plant-based systems but the best production platform should be determined empirically with a case-by-case approach, as described here.

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.

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the ...

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

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is the challenge of identifying suitable vaccine candidates. Malarial proteins expressed using prokaryotic and eukaryotic cell based expression systems are poorly glycosylated, generally insoluble and undergo improper folding leading to reduced immunogenicity. The wheat germ, rabbit reticulocyte lysate and Escherichia coli lysate cell free expression systems are currently used for expression of malarial proteins. However, the length of expression time and improper glycosylation of proteins still remains a challenge. We demonstrate expression of Plasmodium proteins in vitro using HeLa based cell free expression systems, termed &#8220;in vitro human cell free expression systems&#8221;. The 2 HeLa based cell free expression systems transcribe mRNA in 75 min and 3 &#181;l of transcribed mRNA is sufficient to translate proteins in 90 min. The 1-step expression system is a transcription and translation coupled expression system; the transcription and co-translation occurs in 3 hr. The process can also be extended for 6 hr by providing additional energy. In the 2-step expression system, mRNA is first transcribed and then added to the translation mix for protein expression. We describe how to express malaria proteins; a hydrophobic PF3D7_0114100 Maurer&#8217;s Cleft &#8211; 2 transmembrane (PfMC-2TM) protein, a hydrophilic PF3D7_0925900 protein and an armadillo repeats containing protein PF3D7_1361800, using the HeLa based cell free expression system. The proteins are expressed in micro volumes employing 2-step and 1-step expression strategies. An affinity purification method to purify 25 &#181;l of proteins expressed using the in vitro human cell free expression system is also described. Protein yield is determined by Bradford&#8217;s assay and the expressed and purified proteins can be confirmed by western blotting analysis. Expressed recombinant proteins can be used for immunizations, immunoassays and protein sequencing.

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

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.

Malaria causes significant global morbidity and mortality. No routine vaccine is currently available. One of the major reasons for lack of a vaccine is ...

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

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

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.

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...

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

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