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

Summary

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

Abstract

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.

Introduction

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 elimination^2,3. There are five Gzms in humans (GzmA, B, H, K and M), and 10 Gzms in mice (GzmA – 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 PFN^5,6. However, more recent studies also demonstrated extracellular effects of the Gzms with profound impact on immune regulation and inflammation, independently of cytosolic delivery by PFN^7,8. The spectrum of cells that are killed efficiently after cytosolic entry of the Gzms was also recently widened from mammalian cells to bacteria^9,10and 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 lines^12-14. 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 and^12,14). Recombinant Gzms were produced in bacteria¹⁵, yeast¹⁶, insect cells¹⁷and in even in mammalian cells such as HEK 293^18,19. 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 cells^20-22. 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 Β-actin promoter; together, these elements demonstrated the strongest promoter activity in various cell lines²⁴. In addition, the plasmid contains a rabbit Β-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’s Modified Eagle’s Medium (DMEM), and are well suited for cost-efficient calcium-phosphate transfection method.

Protocol

1. Production of the expression plasmid pHLsec-Gzm

1. 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.
2. Confirm correct inserts by sequencing. Expand the expression plasmids in DH5α cells and purify using an endotoxin-free plasmid isolation kit and follow the manufacturer’s instructions.
3. Resuspend the purified plasmids in endotoxin-free, sterile water at a concentration of 2 mg/ml and store at -20 °C until use.

2. Expression of Gzms in HEK293T cells

1. Preparation of reagents
   1. Prepare standard culture medium. To Dulbecco'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 μg/ml).
   2. Prepare transfection medium. To culture medium without penicillin-streptomycin add 25 μg/ml chloroquine (add freshly on the day of transfection from 1,000x stocks in PBS, stored at -20 °C).
   3. Prepare serum-free culture medium: To serum-free medium for HEK293 cells add glutamine (4 mM), penicillin (100 units/ml), streptomycin (100 μg/ml), and 2.5mg/L amphotericin B.
   4. Prepare the solutions described in Table 2 and filter with a 0.45 μm filter before use:
2. Expanding HEK293T cells
   1. Grow HEK293T cells in 20 ml culture medium using 150 x 25 mm tissue culture dishes.
   2. Split cells at 80% confluency (split-ratio of 1:4, usually every 3^rd day). Cells loosely attach, they can be mechanically detached without trypsinization by rigorously pipetting up and down.
   3. 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.
3. Calcium-phosphate transfection
   1. 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 μg of plasmid DNA with 10.95 ml ddH₂0 and 1.55 ml of 2M CaCl₂ in 50 ml tubes (1 tube/5 dishes).
   2. 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.
   3. 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.
   4. Incubate the culture dishes for 7 to 11 hr in a tissue culture incubator (37°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.
   5. Analyze the transfection and expression efficiency by running a sample (~20 μl) of the cell supernatant on SDS-PAGE and staining with Coomassie Brilliant Blue to visualize a granzyme band.
4. Purification of Gzms from culture supernatant by Nickel-IMAC
   1. 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 °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 °C to remove any remaining cell debris.
   2. 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).
   3. Equilibrate a Nickel-IMAC column with His-binding buffer A (10 column volumes, CV) using a suitable FPLC system.
   4. Apply the cleared supernatant to the equilibrated column at a flow rate of 0.5 ml/min at 4 °C.
   5. After the supernatant was applied, wash column with His-binding buffer until UV absorbance baseline is reached (usually 10 CV).
   6. 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.
5. Enterokinase (EK) treatment and final clean-up by cation exchange chromatography
   1. Pool Gzm containing fractions in a dialyzing tube with ≤10 MWCO. Store a small sample at -20 °C as pre-EK control.
   2. 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).
   3. The next day, analyze EK treated Gzms in comparison to the pre-EK control by SDS-PAGE and Coomassie staining.
   4. 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 μm).
   5. 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 °C.
   6. 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.
   7. 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 μM) in spin filters (15 ml, 10 MWCO). Aliquot the concentrated Gzm preparations and store at -80 °C.

3. Testing Gzm activity

1. Preparation of reagents
   1. Prepare colorimetric assay buffer: 50 mM Tris-Base, pH 7.
      1. 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′-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 °C) to the buffer freshly before the assay.
   2. Prepare cleavage assay buffer: 100 mM NaCl, 50 mM Tris-Base, pH 7.5
2. Colorimetric assays
   1. Distribute small Gzm samples (<5 μl) 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).
   2. Add 100 μl of assay buffer to each well (using a multi-channel pipette). Incubate for 10 min at 37 °C. Measure OD at 405 nm in a microplate reader.
3. Cleavage assay
   1. 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.
   2. 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 °C three times. Remove cell debris by centrifugation (15,000 x g for 10 min at 4 °C).
   3. Co-incubate the cleared lysate with Gzms at various concentrations and times as above. Cleavage is detected by immunoblotting using appropriate antibodies.
      ​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 GzmB^27,28; multiple heterogeneous nuclear ribonucleoproteins (hnRNPA1, A2 and U) cleaved by GzmA²⁹; cytomegalovirus phosphoprotein 71 by GzmM³⁰.

Results

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 systems^9,29,31-34.

Discussion

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 bacteria^9,10, 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...

Materials

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