Cell lines and media
The CHO-K1 (ATCC®CCL-61™) and Raji (ATCC®CCL-86™) cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). CHO-K1 cells were grown and maintained at 37°C or 30°C with 70% humidity and 5% CO2 in HAM F12 media (Gibco, Big Cabin, OK, USA) supplemented with 2% fetal bovine serum (FBS, Gibco, Big Cabin, OK, USA) and were used in experiments on protein production. Raji cells were grown and maintained at 37°C with70% humidity and 5% CO2 in RAMP media (Gibco, Big Cabin, OK, USA) supplemented with 10% FBS and were used in FACS direct ligation experiments.
Plasmids and cloning
pCOMIRES HIL anti-CD20 is a tricistronic vector that encodes both the heavy and the light chains of an anti-CD20 antibody along with a neomycin resistance gene under the control of a synthetic CMV promoter. This vector was transfected into CHO-K1 cells to obtain IgG (anti-CD20)-producing cells. The human xbp-1(s) coding sequence was chemically synthesized by GeneScript (Piscataway, NJ, USA). The restriction enzymes Hind III and BamH I (Fermentas, Ontario, Canada) were used to obtain the xbp-1(s) insert and then clone it into the inducible expression plasmid pcDNA™4/TO/myc-His A from the Invitrogen T-REx™ system (Invitrogen, Carlsbad, CA, USA). This plasmid was used to co-transfect IgG-producing stable clones of CHO cells along with the regulatory plasmid pcDNA6/TR (Invitrogen, Carlsbad, CA, USA). To confirm xbp-1(s) cloning, XL1-blue bacterial cells (Stratagene, La Jolla, CA, USA) were transformed with ligated DNA. Ampicillin (Sigma, Ronkonkoma, NY, USA)-selected colonies were isolated and processed for DNA extraction and purification, which was performed using a QIAprep Miniprep Kit (Qiagen, Valencia, CA, USA). Restriction analysis and sequencing (using CMV forward primer 5′-CGCAAATGGGCGGTAGGCGTG-3′ and BGH reverse primer 5′-TAGAAGGCACAGTCGAGG-3′) confirmed the cloning of the xbp-1(s) insert.
Transfection with pCOMIRES anti-CD20 DNA (IgG-encoding plasmid) into CHO cells and generation of stable IgG-producing cells
The transfection of pCOMIRES HIL anti-CD20 plasmid (encoding an anti-CD 20 (IgG) antibody, a secretable protein with molecular weight 150 kDa (two light chains, each with molecular weight 25 kDa, and two heavy chains, each with molecular weight 50 kDa)) into CHO cells was performed using a PolyPlus (JetPrime, New York, NY, USA) kit in six-well test plates (TPP, San Diego, CA, USA) according to the manufacturer’s instructions. The clones harboring the pCOMIRES HIL anti-CD20 transgene were selected from a mixed population by the single-cell dilution method. Geneticin (Roche, Gaillard, France) was used for selection at 800 μg/mL.
Transfection with the T-REx™ -XBP-1(s) system into stable IgG-producing clones of CHO cells and generation of stable double clones (IgG-T-REx-XBP-1(s) cells)
The co-transfection of T-REx-xbp-1(s) plasmid (encoding a spliced form of human apoptotic XBP-1 protein with predicted molecular weight 40 kDa) along with regulatory plasmid pcDNA6/TR into one of the stable IgG-producing clones was performed using a PolyPlus (JetPrime, New York, NY, USA) kit according to the manufacturer’s instructions in six-well test plates (TPP, San Diego, CA, USA). Blasticidin (Sigma, Ronkonkoma, NY, USA) and Zeocin (Sigma, Ronkonkoma, NY, USA) were added to a final concentration of 0.5 μg/mL and 50 μg/mL, respectively. The selective markers encoded by regulatory plasmid pcDNA6/TR and expression plasmid pcDNA™4/TO/myc-His A are against blasticidin and Zeocin, respectively.
Doxycycline induction
Selected IgG-T-REx-XBP-1(s) cells (after the first transfection, IgG clones; after the second, co-transfection, T-REx-XBP-1(s) clones) were induced by DOX at different concentrations: 0 μg/mL for control, 0.1 μg/mL, 0.5 μg/mL and 1 μg/mL. We used these concentrations because we found out that 5 μg/ml and 7.5 μg/ml of doxycycline completely inhibits cells growth for clones and wild type CHO-K1 cells. DOX induction was performed 24 hr after IgG-T-REx-XBP-1(s) cells seeding at a uniform cell density (0.5 × 105 cells/mL) in tissue culture flasks (75 cm2, TPP, San Diego, CA, USA) and then incubated for seven days at 37°C or 30°C. All cultures reached at least 80% under these conditions. Samples were collected for viable cell density, Ab detection by ELISA, nuclear extract isolation and ER staining. Half of the cells in each group continued to grow for seven more days in DOX-free medium after DOX wash-out. Independently, IgG-T-REx-XBP-1(s) cells were incubated for 42 days at 30°C (150 cm2 flasks, TPP, San Diego, CA, USA) with or without 1 μg/mL DOX. In all DOX induction experiments, DOX was added (at an appropriate concentration) every three days to the cell culture. Induction experiments were performed twice in duplicate (four independent culture samples per group).
Viability assay
The viable cell density of the IgG-T-REx-XBP-1(s) cells were tested under different DOX concentrations (0 μg/mL, 0.1 μg/mL, 0.5 μg/mL or 1 μg/mL) every day during seven days of cell growth at 37°C and 30°C. Seeding was performed at a uniform cell density (0.06 × 105 cells/mL) in six-well tissue culture plates (TPP, San Diego, CA, USA). At the seventh day all cultures reached at least 80% under these conditions. In addition, the viable cell density of IgG-T-REx-XBP-1(s) cells was tested on the seventh day of growth with DOX and on the seventh day after wash-out in DOX-free medium. In addition, IgG-T-REx-XBP-1(s) cells were tested every seventh day during 42 days of cell growth under 1 μg/mL DOX (or 0 μg/mL as control) at 30°C. The viable cell density was measured using the trypan blue (Sigma, Ronkonkoma, NY, USA) exclusion method with a hemocytometer and light microscope for manual cell counting. Every viable cell density experiment was performed twice in duplicate (single determination from each of two independent culture samples per group in two independent experiments).
ELISA
The supernatants of IgG-T-REx-XBP-1(s) cells in the presence or absence of DOX were collected every seventh day of 37°C or 30°C growth for two weeks or every seventh day for six weeks and processed for analysis by enzyme-linked immunosorbent assay (ELISA) (duplicate determination from each of two independent culture samples per group in two independent experiments). The Lunc/Maxisorp Immunoplate (Thermo Scientific, Waltham, MA, USA) was incubated with primary antibody (goat anti-human IgG (H + L), 1:3000 dilution; Thermo Scientific, Waltham, MA, USA) and blocked with 3% fat-free dehydrated milk solution. After blocking and washing the plate, the supernatants were applied to the plate and incubated for 2 hr. The plate was washed again, and secondary antibody (anti-human IgG Fc-specific, alkaline phosphatase-conjugated, produced in goat, 1:3000 dilution; Sigma, Ronkonkoma, NY, USA) was applied for 1 hr. The plate was washed again, and at the end of the procedure, the signal of absorbance was read at 405 nm by a microplate reader (ELx800 96-well Microplate Reader, MTX Lab Systems, Inc., Vienna, VA, USA) after 4-Nitrophenyl phosphate disodium salt solution (pNPP) (Invitrogen, Carlsbad, CA, USA) addition. In addition, human IgG (whole molecule; Thermo Scientific, Waltham, MA, USA) was used in different concentrations as a control on the same plate.
Isolation and purification of produced proteins
The IgG produced under different temperature conditions by IgG-T-REx-XBP-1(s) cells was purified on the HiTrap™ Protein A HP 1 mL (GE Life Sciences, Pittsburgh, PA, USA) column. The column was first equilibrated with 10 mL Protein A IgG Binding Buffer (Thermo Scientific, Waltham, MA, USA) at a rate of 1 mL/min. Then, the supernatant from IgG-T-REx-XBP-1(s) cells was applied to the equilibrated column. The column was washed with 30 mL Protein A IgG Binding Buffer (Thermo Scientific, Waltham, MA, USA). Then, the protein was eluted with 50 mL IgG Elution Buffer (Thermo Scientific, Waltham, MA, USA), and 2 mL per fraction was collected. Fractions were neutralized with Tris–HCl pH 9.0. The Ab present in the fractions was immunodetected in a dot blot assay. Five microliters of each fraction was directly pipetted onto a nitrocellulose Hybond-C Extra membrane (Amersham® Bioscience, Piscataway, NJ, USA). The membrane was blocked with 3% fat-free milk solution and incubated with anti-human IgG (Fc-specific, alkaline phosphatase-conjugated, produced in goat, 1:3000 dilution) (Sigma, Ronkonkoma, NY, USA), and the proteins were revealed using a BCIP/NBT substrate Kit (Invitrogen, Carlsbad, CA, USA). The Ab-containing fractions were selected for dialysis, which was performed using a Centricon YM-50 (Amicon Bioseparations, Billerica, MA, USA) in PBS buffer (10 mM NaH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4).
Western blotting
Anti-CD20 antibody was also detected by western blotting. Five hundred nanograms of IgG sample was loaded in each well of a Bis-Tris gel (NuPAGE® Novex 4-12% Bis-Tris Gel, Invitrogen, Carlsbad, CA, USA) and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) according to the manufacturer’s instructions. The proteins were transferred to the Hybond-C Extra nitrocellulose membrane (Amersham® Bioscience, Piscataway, NJ, USA) and blocked in 3% fat-free milk PBS solution. The immunodetection was performed using anti-human IgG (Fc-specific, alkaline phosphatase-conjugated, produced in goat (1:3000 dilution) (Sigma, Ronkonkoma, NY, USA) with a BCIP/NBT substrate Kit™ (Invitrogen, Carlsbad, CA, USA).
XBP-1(s) was also probed by western blotting. The nuclear extracts from the IgG-T-REx-XBP-1(s) cells were prepared as described by Becker and colleagues [10]. Briefly, the nuclear extracts were prepared from 5×106 cells/per sample and equal volumes of nuclear extracts were loaded into a Bis-Tris gel (NuPAGE® Novex 4-12% Bis-Tris Gel, Invitrogen, Carlsbad, CA, USA), and SDS-PAGE was performed according to the manufacturer’s instructions. Samples were transferred to the Hybond-C Extra nitrocellulose membrane (Amersham® Bioscience, Piscataway, NJ, USA), and after blocking with 3% fat-free milk PBS solution, rabbit anti-human-XBP-1(s) (1:1000 dilution; Sigma, Ronkonkoma, NY, USA) was added, followed by alkaline phosphatase-conjugated anti-rabbit IgG incubation (1:1000 dilution; Sigma, Ronkonkoma, NY, USA). The proteins were revealed using a BCIP/NBT substrate Kit™ (Invitrogen, Carlsbad, CA, USA).
Fluorescence-activated cell sorting (FACS)-ER staining
The IgG-T-REx-XBP-1(s) cells that were grown for seven days under DOX induction and those that were grown for one more week after wash-out were collected at 3×105 cells/per staining and washed with HBSS buffer (140 mM NaCl, 4.7 mM KCl, 1 mM MgCl2, 1.5 mM CaCl2, 10 mM glucose, 10 mM HEPES, pH 7.4). After washing with HBSS buffer, the cells were labeled with 250 nM of ER-Tracker™ Green Dye (ER-Tracker™ Green Dye for Live-Cell Endoplasmic Reticulum, Molecular Probes, Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s manual. The samples were washed again with HBSS buffer and analyzed using a BD FACS Verse flow cytometer (BD Bioscience, San Jose, CA, USA). Ten thousand events were collected per sample using no gate for acquisition. The dead cells were not excluded in the analysis. We used BD FACSuite to data acquisition. The experiment was performed twice in duplicate.
FACS direct ligation assay
Raji cells were grown for five passages as described above, collected and resuspended in 1 part RAMP media with 10% FBS and 1 part FACS buffer (PBS supplemented with 2% FBS) at 3×106 cells/well in a 96-well plate (TPP, San Diego, CA, USA). After centrifugation, the cells were blocked with FcR blocking reagent (MACS, Biotec, Bergisch Gladbach, Germany) on ice for 30 minutes according to the manufacturer’s instructions. Purified and dialyzed IgG samples, which were produced by IgG-T-REx-XBP-1(s) cells at 37°C and 30°C, and commercial IgG (rituximab, MabThera, Genetech Inc., South San Francisco, CA, USA) as a positive control were added at 100 ng per well. Samples were incubated on ice for 1 hr and centrifuged after the addition of FACS buffer. The cells were washed twice with FACS buffer and incubated with mouse FITC anti-human IgG (BD Pharmingen™, BD Biosciences, San Jose, CA, USA) according to the manufacturer’s manual. The cells were incubated on ice for 30 minutes in the dark, washed again twice with FACS buffer and processed for fluorescence intensity measurements using a BD FACS Verse flow cytometer (BD Bioscience, San Jose, CA, USA). Each experiment was performed twice in duplicate.