Cells and antibodies
The competent E. coli strain BL21 (F-ompT hsdS (rB-mB-) gal dcm; providded by aTyr Pharma) was used as the host for rhTyrRS expression. This strain was transformed with the pET24a inducible expression vector in which the His-tag sequence was deleted and the T7 promoter was replaced with a Tac promoter.
A mouse anti-human IL-8 monoclonal antibody (BC013615, Proteintech Group), rabbit anti-human IL-8 polyclonal antibody (BC013615, Proteintech Group), and goat anti-rabbit IgG-HRP SC-2004 antibody (D2111, Santa Cruz Biotechnology) were used.
Growth conditions
A clone with a high rhTyrRS expression level was cultivated in LB agar and M9CA medium (10 g glucose, 6 g Na2HPO4, 35 g KH2PO4, 2.93 g NaCl, 0.4 g NH4Cl, 1.2 g MgSO4, and 1 mL trace element solution per liter). Trace element solution (1 L) consisted of 2.8 g FeSO4 · 7H2O, 2 g MnCl2 · 4H2O, 2.8 g CoSO4 · 7H2O, 1.5 g CaCl2 · 2H2O, 0.2 g CuCl2 · 2H2O, and 0.3 g ZnSO4 · 7H2O. Feeding solutions were 50% glycerol, 250 g/L glucose, and 100 g/L yeast extract. The glucose and MgSO4 solutions were sterilized separately. Kanamycin sulfate was added to a final concentration of 100 μg/mL in both the M9CA and feeding solutions.
Fermentation was performed in a 5-L Bioflo 3000 fermenter (New Brunswick Scientific, New Brunswick, NJ, USA) with automated control of: the pH at 7.0 by the addition of ammonium hydroxide, dissolved O2 at 70% by providing pure oxygen, and agitation at 700 rpm. Air was provided at a flow rate of 4.0 L/min and the temperature was controlled at 30°C. Fermentation was conducted according to the process developed by Shiloach et al. (1996) [17]. The batch phase ends when cells have used up the available glucose. The best indications that the batch phase has ended include a sharp decrease in stirrer speed and an increase in pO2. The bacterial concentration was measured off-line by the optical density at 600 nm and induced with 0.5 mM isopropylthio-β-galactoside (IPTG) once it reached an OD600 of 30 (~10 h). After 6 h of 0.5 mM IPTG induction, the cells were harvested by centrifugation at 6,000 × g for 30 min. The cell pellet was stored at -70°C.
Cell lysis
Harvested cell pellets were resuspended in 10 volumes of 20 mmol/L HAc-NaAc buffer (pH 6.0) and subjected to two cycles of microfluidization at 1000 bar. The crude extract was then centrifuged at 10,000 × g for 60 min.
Cation exchange chromatography
The clarified supernatant was loaded onto a SP Sepharose Fast Flow column (GE) that was pre-equilibrated with 20 mmol/L HAc-NaAc buffer (pH 6.0). The bound proteins were eluted with a linear NaCl gradient (0 to 1 mol/L). Fractions containing rhTyrRS were pooled and analyzed by SDS-PAGE.
Gel filtration
The pooled fractions were loaded onto a Sephadex-G50 column (GE) pre-equilibrated with 20 mmol/L phosphate buffer (PB; pH 7.0) and rhTyrRS was washed with 20 mmol/L PB at 5 mL/min. Fractions were collected from the column and analyzed by SDS-PAGE.
Anion exchange chromatography
The diluted product solution was loaded onto a 50-mL Q Sepharose Fast Flow column (GE) pre-equilibrated with 20 mmol/L PB (pH 7.0). RhTyrRS was eluted with a linear NaCl gradient (0 to 1 mol/L) and identified by SDS-PAGE via Coomassie blue staining.
SDS-PAGE and sequencing of the N-terminal amino acids
Electrophoresis was carried out in 1-mm-thick gels using BioRad MiniGel apparatus. Coomassie staining was performed as previously described, except that microwave heating was used at each staining step to reduce the total staining and destaining procedure time to 30 min. The protein concentration was measured using BSA as a standard.
The sequence of the N-terminal amino acids of purified rhTyrRS was determined using a protein sequencer (PPSQ-33A, USA).
Western blotting
Proteins resolved in a pre-cast Bis-Tris gel (BioRad) were electrotransferred to a PVDF membrane followed by blocking in 10% BSA solution prepared in TBST (Tris-buffered saline with 0.1% Tween-20). The membrane was then incubated with anti-rhTyrRS monoclonal antibody (1/5000) for 90 min at room temperature. After washing, the membrane was incubated with peroxidase-conjugated goat anti-mouse IgG (1/100,000) for 60 min at room temperature. All antibody incubations and washing steps were carried out in TBST. The immunoreactive bands were visualized with a Western Blot kit (Thermo 34077).
Reversed-phase HPLC
Reversed-phase HPLC was used to study the purity of rhTyrRS. A Higgins Proto 300 C4 column (Waters 150 × 4.6 mm) was used at a flow rate of 1 mL/min and at a temperature of 40°C. The column was eluted with a linear 30-min gradient from 5% B to 95% B (A = 0.1% Trifluoroacetic Acid (TFA) in HPLC water; B = 0.1% TFA in acetonitrile) and monitored by absorbance at 215 nm with a total run time of 40 min.
Mass spectrometry analysis
The purified protein was analyzed by mass spectrometry. Molecular weight measurements were made by LC-MS with a quadrupole-time-of-flight mass spectrometer at the Fudan University Institute of Biomedical Science.
Aminoacylation assay
Aminoacylation activity was determined at ambient temperature in 150 mmol/L Tris–HCl (pH 7.5), 150 mmol/L KCl, 10 mmol/L MgCl2, 10 mmol/L β-mercaptoethanol, 4 mmol/L ATP, and 10 mmol/L tyrosine (including 3 mmol/L [3H]-tyrosine, GE Healthcare). Human tRNATyr was prepared as previously described [12]. Before each assay, 100 mmol/L human tRNATyr was annealed by heating at 65°C for 5 min and cooled to room temperature. Reactions were initiated by the addition of rhTyrRS (10 nmol/L) to the reaction mixture. Aliquots were taken at fixed intervals and spotted onto Whatman filter discs saturated with 5% trichloroacetic acid and dried. The filters were placed into ice-cold 5% trichloroacetic acid and washed three times with fresh 5% trichloroacetic acid at 4°C and once with 95% ethanol. The level of tRNA aminoacylation was then quantitated by liquid scintillation counting.
Biological activity assay
RhTyrRS activity was tested using ELISA to measure IL-8 secretion by rhTyrRS-treated HL-60 cells. Cells were thawed from vials and cultured according to the American Type Culture Collection instructions, except that RPMI-1640 (ATCC 30–2001) was supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies, formerly Invitrogen 10082–147) and used as growth medium. Cells were plated in a 96-well plate at a density of 1 × 106 cells per 1 mL of medium on the day of the assay. The rhTyrRS was added immediately after cell plating. Cells were plated at 108 μl-1 per well. The protein was diluted in sterile phosphate-buffered saline (PBS; Life Technologies, formerly Invitrogen 10010–023) to a concentration of 10 μmol/L (10× protein stock). The 10× protein stock was serially diluted and 12 μL was added to each well such that the final protein concentrations were 500 nmol/L, 250 nmol/L, 125 nmol/L, 62.5 nmol/L, 31.25 nmol/L, and 15.625 nmol/L. PBS was used as a control. Cells were incubated at 37°C and 5% CO2 for 24 h. The 96-well plate was then spun for 10 min at room temperature in a swinging bucket centrifuge at 1,000 × g. The plate was tilted at a 45° angle and 100 μL of media per well was transferred to a new 96-well plate. The amount of secreted IL-8 protein in the media was then determined by sandwich ELISA.
