Human colon carcinoma cell line HCT116 was obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained according to the ATCC’s instructions. Human primary proliferating cardiac fibroblasts were obtained from Cell Applications, Inc. (San Diego, CA). Briefly, HCT116 cells were cultured in McCoy’s 5A medium, supplemented with 10% fetal bovine serum (FBS) and gentamicin (50 µg/ml) (all from Gibco, Paisley, UK) in a 37 °C humidified atmosphere with 5% CO2. Human cardiac fibroblasts were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma Aldrich, Saint Louis, MI) supplemented with 10% FBS and 1:100 penicillin/streptomycin (all from Life Technologies, Carlsbad, CA) in a 37 °C humidified atmosphere with 5% CO2. The cells were regularly tested for Mycoplasma sp. contamination by PCR-ELISA kit (Roche, Mannheim, Germany) and for endotoxin contamination by the Limulus test (Charles River Laboratories, Wilmington, MA) according to manufacturer’s instruction.
Bacterial cell cultures
Lactococcus lactis NZ9000 host strain, a derivate of L. lactis subsp. cremoris MG1363 with regulatory genes (nisR, nisK) integrated into the pepN gene of MG1363 , was obtained from MoBiTec (Goettingen, Germany) and cultured in M17 medium (BTL, Lodz, Poland) supplemented with 0.5% glucose (POCH, Gliwice, Poland). For culture of L. lactis clones harbouring secretion plasmid vector pNZ8124 (MoBiTec) and its modified derivatives with the human soluble TRAIL-cDNA placed downstream of the inducible promoter PnisA on the plasmid pNZ8124, chloramphenicol (10 µg/ml; Sigma Aldrich) was added to maintain the plasmid.
A synthetic human soluble TRAIL-cDNA (hsTRAIL-cDNA) with optimized codons was designed to fit the codon usage pattern (codon bias) of the L. lactis host. This synthetic cDNA construct contained 169 codons plus stop codon, including 76 original and 94 changed codons for those more frequently found in L. lactis highly expressed genes. Synthetic hsTRAIL-cDNA construct was commercially synthetized by Eurofins Genomic (Ebersberg, Germany). Then, constructed hsTRAIL-cDNA sequence was ligated, using T4 DNA ligase (EurX, Gdansk, Poland), to EcoRV and XbaI—linearized plasmid vector pN8124 (MobiTec), containing sequence coding for signal peptide of lactococcal usp45 gene. Prepared pNZ8124-hsTRAIL plasmid vector was transformed into the electrocompetent L. lactis NZ9000 host strain cells by electroporation, using Gene PulserXcell™ Electroporation System (BioRad, Hercules, CA), according to vector producer’s instruction (MoBiTec).
Selection of a positive clone of L. lactis harbouring recombinant plasmid pNZ8124-hsTRAIL
Positive clone of L. lactis containing insert for hsTRAIL-cDNA after electroporation with plasmid vector pNZ8124-hsTRAIL was selected in two-steps. First, isolated plasmids were cleaved with EcoRV and XbaI restriction enzymes (EurX) and the presence of hsTRAIL-cDNA insert was defined by the size of cleaved fragments using agarose gel electrophoresis (1.5% agarose gel). In the second step, selected fragments were purified from the gel and amplified by PCR method using the following primers: 5′-TGGTACTCGTGGTCGTAGCA-3′ sense and 5′-GAAGCTTCGTGGTCCATGTC-3′ antisense (Genomed, Warsaw, Poland). Clone number 3 was selected as TRAIL-positive, designated as L. lactis (hsTRAIL+) and used for further studies.
Optimization of culture conditions for the recombinant L. lactis (hsTRAIL+) clone
M17 broth medium (BD Difco, Franklin Lakes, NJ) supplemented with 0.5% glucose (Gluc) and 10 µg/ml of chloramphenicol (Cm10), was inoculated with L. lactis (hsTRAIL+) glycerol stock and grown overnight at 30 °C, without aeration. To optimize conditions for culture of the recombinant L. lactis clone, the following culture media were prepared: M17 supplemented with 0.5% Gluc, Cm10; M17 supplemented with 0.1% Gluc, 0.1% of l-arginine (Arg) and Cm10; M17 supplemented with 0.3% Gluc, 0.3% Arg and Cm10, and then were inoculated with an overnight pre-culture of bacteria in a dilution of 1:20, and incubated at 30 °C, without aeration. The OD600 and pH of the cell cultures were determined after 4 and 24 h. A 4-h culture period was selected and further used for the production of hsTRAIL by L. lactis producer (clone no. 3) upon induction with nisin.
Induction of hsTRAIL expression with nisin
For induction of hsTRAIL expression and secretion by L. lactis (hsTRAIL+) bacteria, the M17 broth medium supplemented with 0.5% Gluc and Cm10 was inoculated with L. lactis (hsTRAIL+) or L. lactis with empty vector pNZ8124—L. lactis (hsTRAIL−)—used as a negative control. Cultures were diluted 1:40 and grown overnight at 30 °C, without aeration. Overnight cultures were diluted 1:20 in M17 broth medium supplemented with 0.3% Gluc, 0.3% Arg, Cm10 and ZnSO4 (100 µM) and grown for additional 3 h at 30 °C without aeration until OD600 = 0.3–0.4. After incubation, the cultures were centrifuged for 30 min at 2800×g at room temperature and the cell pellets were resuspended in 1/4 volume of M17 supplemented with 0.3% Gluc, 0.3% Arg, Cm10, ZnSO4 (100 µM) and aprotynin (BioShop, Burlington, Canada), as the serine proteases inhibitor, to prevent potential cleavage of expressed hsTRAIL protein, and were induced for hsTRAIL expression with nisin (MoBiTec). For optimization of induction conditions, the cultures were induced with the following concentrations of nisin: 10; 25; 35; 50; 80 ng/ml in M17 medium supplemented as above. The optimal concentration of aprotynin was established experimentally from 2 to 5 µg/ml tested. After 4 h of incubation, the OD600 of the cultures was measured to monitor the bacterial growth and then the cells were centrifuged for 30 min at 2800×g at 4 °C. Cell-free supernatants were collected, pH was measured and neutralized (if necessary) to pH = 7 with NaOH (POCH, Gliwice, Poland). TRAIL samples concentration was performed using disposable The Thermo Scientific™ Pierce™ PES 10 K protein concentrators (Pierce Biotechnology, Rockford, IL) for centrifugal ultrafiltration, according to the manufacturer’s instructions. Concentrated supernatants were filtered through low protein-binding filters (Merck-Millipore, Burlington, MA), aliquoted and stored at − 80 °C until use for further studies.
Isolation of hsTRAIL protein
hsTRAIL was precipitated as total protein content from sterile culture supernatants using chloroform–methanol protein extraction method . Briefly, 600 µl of methanol was added to 150 µl of culture supernatant and vortexed, after which chloroform, at ratio 1:1 to the starting volume of the supernatant, was added and vortexed again. Next, 450 µl of H2O was added, and the whole suspension was vortexed and centrifuged for 5 min at 14,000×g. The top, aqueous layer was removed, then 600 µl of methanol was added and the mixture was vortexed and centrifuged for 10 min at 14,000×g. Methanol was removed and the pellet was dried under vacuum for 1.5 h, and resuspended in Bacterial Protein Extraction Reagent (BPER, Thermo Fisher Scientific, Waltham, MA) with addition of protease inhibitor cocktail (Pierce, Waltham, MA) and stored at − 20 °C until further use.
Western blot analysis of hsTRAIL produced by L. lactis (hsTRAIL+) bacteria
The protein samples precipitated from supernatants of L. lactis (hsTRAIL±) cultures were used for protein separation by the SDS-PAGE electrophoresis and Western blot analysis. Protein concentration was measured using BCA (Bicinchoninic acid) protein assay kit (Pierce) and the equal amounts of the samples were mixed with LDS sample buffer (lithium dodecyl sulfate at pH of 8.4, Invitrogen, Carlsbad, CA) and reducing buffer (50 mM dithiothreitol—DTT; Invitrogen), incubated at 70 °C for 10 min and loaded onto 14% SDS-PAGE gel. Recombinant human TRAIL (rhTRAIL; PeproTech, London, UK) was used as a positive control. After electrophoresis, separated proteins were transferred onto the polyvinylidene fluoride membrane (PVDF, BioRad) using Trans-Blot Turbo Transfer System (BioRad). Subsequently blots were blocked for 1 h at room temperature with 5% of nonfat milk in TTBS buffer (50 mM Tris–HCl, pH 7.6; 150 mM NaCl, 1% Tween-20). The protein bands were detected using the following antibodies: primary—mouse anti-human sTRAIL/Apo2L monoclonal antibodies (dilution 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA); secondary—goat anti-mouse HRP-conjugated IgG (dilution 1:8000; Santa Cruz Biotechnology) and visualized with the SuperSignal West Pico Chemiluminescence Substrate kit (Pierce) according to the manufacturer’s protocol and analysed with KODAK GEL LOGIC 1500 Digital Imaging System (KODAK, Rochester, NY).
SYPRO® Ruby Protein gel staining
The protein samples precipitated from supernatants of L. lactis (hsTRAIL±) cultures, or crude supernatants samples, were used for protein separation by the SDS-PAGE electrophoresis. Protein concentration was measured using BCA (Bicinchoninic acid) protein assay kit (Pierce) and the equal amounts of the samples were mixed with LDS sample buffer (lithium dodecyl sulfate at pH of 8.4, Invitrogen, Carlsbad, CA) and reducing buffer (50 mM dithiothreitol—DTT; Invitrogen), incubated at 70 °C for 10 min and loaded onto 14% SDS-PAGE gel. After electrophoresis, SYPRO® Ruby Protein gel staining (Molecular Probes, Eugene, US) was performed to detect proteins present in supernatants. The gel was fixed (50% methanol, 7% acetic acid) for 30 min, stained overnight with SYPRO® Ruby gel stain, washed in wash solution (10% methanol, 7% acetic acid) for 30 min. and rinse twice in ultrapure water. The gel was analyzed using ChemiDoc™ Imaging system (BioRad).
Sequencing TRAIL protein by mass spectrometry (LC–MS/MS)
The visualized SDS-PAGE bands (Coomassie Brilliant Blue staining) were cut out and proteins were reduced, alkylated, and digested according to the protocol described previously . Peptides were analyzed with the use of a Q-Exactive mass spectrometer (Thermo Fisher Scientific) coupled with nano-HPLC (UltiMate 3000 RSLCnano System, Thermo Fisher Scientific) as previously described with minor modifications . Peptides were separated using a 90 min gradient of acetonitrile from 2 to 40% in the presence of 0.05% formic acid. The Top 8 method was used for mass spectrometry measurement with full MS and MS/MS resolution of 70,000 and 35,000 respectively. Database searching of RAW files was performed in Proteome Discoverer 1.4 (Thermo Fisher Scientific) MASCOT 2.5.1 (Matrix Science Ltd, London, UK) was used for database searching against the common Repository of Adventitious Proteins (cRAP) database containing the sequences of recombinant tumor necrosis factor ligand superfamily member 10 (TRAIL), lactococcal protein usp45 and common contaminants. The following search parameters were applied: enzyme specificity—trypsin; permitted number of missed cleavages—1; fixed modification—carbamidomethylation (C); variable modifications—oxidation (M), deamidation (NQ); precursor mass tolerance—± 10 ppm; fragment mass tolerance—± 20 mmu. Identifications with a score value over 80 were accepted.
Assessment of TRAIL secretion efficacy
Induction of hsTRAIL secretion was performed as described above. The L. lactis cells in broth culture were centrifuged for 30 min at 2800×g at 4 °C and extraction of protein from bacterial cells pellet was performed. The cells were resuspended in Bacterial Protein Extraction Reagent (BPER, Thermo Fisher Scientific) with addition of protease inhibitor cocktail (Pierce), 10 U of DNase-I and 5.6 mg/ml of lysozyme (both from Thermo Fisher Scientific), then incubated for 1 h at 37 °C and 10 min. at 70 °C to inhibit the enzymes. After centrifugation (10 min., 15,000×g, RT) cell lysates were collected and stored at − 80 °C until further use. To assess the concentration of hsTRAIL in the lysate from recombinant L. lactis (hsTRAIL+) bacteria and secreted to the broth culture medium during induction, ELISA for human soluble TRAIL (LSBio™, Seattle, WA) was performed according to the manufacturer’s instructions. The absorbance was measured at 450 nm and 570 nm (wavelength correction) using microplate reader ELx 800NB (BIO-TEK INSTRUMENTS, Winooski, VT).
Cell viability assay
Cytotoxic activity of hsTRAIL from the culture supernatant of L. lactis (hsTRAIL+) against human colon cancer HCT116 cells and human cardiac fibroblasts was determined using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega, Madison, WI). Briefly, HCT116 cells and human cardiac fibroblasts were seeded onto flat-bottom 96-well plates (Sarstedt, Numbrecht, Germany) at a density of 104/5 × 103 cells per well in McCoy’s 5A/DMEM medium, respectively, containing 2% FBS. After 20 h for cell attachment, the supernatant from the culture of L. lactis (hsTRAIL+) was added to the cells in the dilutions corresponding to the concentrations of hsTRAIL: 25; 50; 75; 100 ng/ml, respectively (measured using ELISA Kit (LSBio™, Seattle, WA). As a negative control, the supernatant from the cultures of L. lactis (hsTRAIL−) was added to the cells in corresponding volumes. As a positive control, recombinant human TRAIL (rhTRAIL; PeproTech) was used in the same range of concentrations. After 48 h of incubation 20 µl per well of MTS (CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega) dye solution was added directly into the culture wells and incubated for additional 2 h. The quantity of formazan product, directly proportional to the number of living cells in culture, was detected by absorbance measurement at 490 nm with a 96-well plate reader (Spark® Tecan, Mannedorf, Switzerland).
Detection of apoptosis by flow cytometry
Apoptosis of HCT116 cells was determined using FITC-Annexin V Apoptosis Detection kit (BD Pharmingen, New Jersey, US) according to the to the manufacturer’s instructions. Briefly, HCT116 cells were seeded onto flat-bottom 24-well plate (Sarstedt) at a density of 105 cells per well in McCoy’s 5A medium containing 2% FBS. After 20 h for cell attachment, the supernatant from the culture of L. lactis (hsTRAIL+) was added to the cells in the dilutions corresponding to the concentration of hsTRAIL 100 ng/ml. As a negative control, the supernatant from the cultures of L. lactis (hsTRAIL−) was added to the cells in corresponding volume. As a positive control, recombinant human TRAIL (rhTRAIL; PeproTech) was used in the same concentration. After 48 h of incubation, the cells were washed twice in ice-cold PBS, trypsynized and cell pellets were respuspended in 1× Annexin V Binding Buffer (0.01 M Hepes/NaOH (pH 7.4), 0.14 M NaCl, 2,5 mM CaCl2). The cells were stained with Annexin V-FITC and PI for 15 min at RT in the dark, followed by FACS analysis using FACSCalibur (Becton–Dickinson Immunocytometry System, Palo Alto, CA) by using CellQuest (version 3.1) software.
Statistical analysis was performed using GraphPad Prism Software version 4.00 (2003). Statistical significance was calculated using two-way ANOVA with Tukey’s multiple comparisons post-test. *p < 0.05, **p < 0.01, ***p < 0.001. The data from each assay are representative for 3–5 independent experiments.