Strains and plasmids
The E. coli strains BL21(DE3) and C41(DE3) were purchased from Novagen® (EMD Biosciences, Inc., Madison, WI, USA) and Lucigen Corporation (Middleton, WI, USA), respectively. The PCR-Blunt® cloning vector was purchased from Invitrogen® (Carlsbad, CA, USA) and the pET-30a (+) expression vector was from Novagen®.
Molecular cloning of rhANXA5
Oligonucleotides were designed based on the human ANXA5 coding sequence in the GenBank (accession number: NM_001154.3) National Institute of Health (NIH) genetic sequence database . Specific primers were designed to contain NdeI (forward primer: 5’ GCG CAT ATG GCA CAG GTT CTC AGA GGC ACT 3’) and HindIII (reverse primer: 5’ GCG AAG CTT TTA GTC ATC TTC TCC ACA GAG C 3’) restriction sites (italics). The human ANXA5 gene sequence was PCR-amplified from a human blood cDNA sample.
The amplified ANXA5 coding sequence was cloned into the PCR-Blunt® vector, cleaved with NdeI and HindIII restriction endonucleases (New England BioLabs®, Ipswich, MA, USA) and subcloned into the pET-30a (+) expression vector, previously digested with the same restriction enzymes, to generate the pET-30a (+)::ANXA5 plasmid. The cloned ANXA5 sequence was confirmed by automated DNA sequencing.
Lysogeny broth (LB) (tryptone, 10 g L−1; yeast extract, 5 g L−1; NaCl, 10 g L−1) was sterilized by autoclaving (30 min at 121°C). LB medium was used for shake-flask cultivation and for inoculum development of bioreactor cultivations.
Semi-defined medium (SD)  was used for shake-flask cultivations as well as fed-batch bioreactor cultivations, as both initial batch and also feeding media (with varied glucose and MgSO4 concentrations). SD medium contains 0.5 g L−1 NaCl (a), 1 g L−1 NH4Cl (b), 20 g L−1 yeast extract (c), 6 g L−1 Na2HPO4 (d), 3 g L−1 KH2PO4 (e), 1 μg L−1 thiamine (f), 1 mM MgSO4 (g), 0.1% trace solution (h), and 0.1 mM CaCl2 (i). For both shake-flask and bioreactor fed-batch cultivations (initial batch culture), SD medium was supplemented with glucose to a final concentration of 5 g L−1 (j). Components (a) to (e) were assembled and sterilized together by autoclaving (30 min at 121°C), while components (g) to (i) were sterilized separately by autoclaving and added under aseptic conditions. Thiamine solution (f) was filter-sterilized. Trace solution (h) contained 2.8 g L−1 FeSO4, 2 g L−1 MnCl2, 2 g L−1 CaCl2, 0.26 g L−1 CuCl2, and 0.3 g L−1 ZnSO4. For feeding medium in bioreactor cultivations, SD medium was supplemented with glucose (final concentration of 300 g L−1) and MgSO4 (40 mM). To prepare this feeding medium, we mixed equal parts of a 2x concentrated SD medium and a 600 g/L glucose stock solution, both sterilized separately. SD medium used in bioreactor cultivations was also supplemented with 100 μL of Antifoam 204 (Sigma-Aldrich, São Paulo, SP, Brazil) per liter of culture. Both LB and SD media were supplemented aseptically with filter-sterilized kanamycin to a final concentration of 30 μg mL−1.
E. coli BL21(DE3) and C41(DE3) strains were transformed with the pET-30a (+) vector or the pET-30a (+)::ANXA5 recombinant plasmid by electroporation. Transformant colonies were selected on LB agar plates containing 30 μg mL−1 kanamycin . Isolated colonies were selected and grown overnight (at 37°C and 180 rpm) in 5 mL of LB supplemented with 30 μg mL−1 kanamycin. To compare the expression profile with different strains (BL21(DE) or C41(DE3)) or media (LB or SD medium), saturated cultures were inoculated in 50 mL of LB or SD medium for shake-flask cultivations and grown at 37°C and 180 rpm to an optical density (OD600 nm) of 0.4–0.6. At this growth stage, we induced rhANXA5 expression by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. At 3, 6, 9 or 24 h after induction, cells were harvested by centrifugation (11,800 g) for 30 min at 4°C, and the pellet was stored at −20°C. The expression of the recombinant soluble protein was confirmed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) visualized by Coomassie® Brilliant Blue R-250 staining. For biomass measurements from shake-flask cultures, isolated colonies were selected and grown overnight (at 37°C and 180 rpm) in 10 mL of LB supplemented with 30 μg mL−1 kanamycin and inoculated in 500 mL of SD medium. Inoculated cultures were grown (37°C and 180 rpm) to an optical density (OD600 nm) of 0.4–0.6, rhANXA5 expression was induced by the addition of IPTG to a final concentration of 1 mM, and cells were harvested after 6 h of IPTG induction.
Inoculum development for bioreactor cultivation
A master cell bank (MCB) of transformed E. coli BL21(DE3) cells containing the pET-30a (+)::ANXA5 recombinant plasmid was prepared in 50% glycerol and stored at −80°C. For inoculum development, 150 μL of E. coli BL21(DE3) MCB cells (stored at −80°C) were grown overnight at 180 rpm and 37°C in 1 L flasks containing 250 mL of LB medium supplemented with 30 μg mL−1 kanamycin. The final optical density (OD600) for each pre-inoculum culture was determined spectrophotometrically. For each experiment, we calculated and collected the initial volume of pre-inoculum culture needed to start bioreactor cultivation with an initial OD600 of 0.1. Collected aliquots were diluted in LB medium to a final volume of 100 mL before inoculation in 900 mL of SD medium for bioreactor cultivation.
Batch and fed-batch culture experiments were conducted in a BIOSTAT® B Plus bioreactor (Sartorius Stedim, Goettingen, Germany) with two 2 L stirred tanks, filled with 1 L of SD medium each, at 37°C, pH 7.0 and supplemented with kanamycin. For pH control, 12% (v/v) ammonium hydroxide and 10% (v/v) phosphoric acid were employed. The bioreactor was equipped with two Rushton turbines and with agitation, aeration, temperature and pH controllers. A polarographic electrode was used to measure the dissolved oxygen concentration (DOC) in the culture. The pO2, pH, stirrer speed, base and acid consumption and aeration rate were measured online and recorded by an external data acquisition and control system (Sartorius Stedim). Feeding was implemented using the bioreactor proprietary software micro-DCU system v. 0.63 (Sartorius Stedim) that allows controlling a peristaltic pump for feeding medium addition. The flow rate varied linearly from 0.066 mL min−1 (starting after 4 h of batch culture) to 0.594 mL min−1 after 26 h of feeding (30 h of bioreactor cultivation). In batch cultures, the DOC was maintained at 30% by cascading agitation (400–1000 rpm) with constant aeration rate (1vvm), and the process was finished when the biomass reached stationary phase.
Fed-batch cultivations were started as batch cultures with feeding starting at 4 h of cultivation (approximately OD600nm 16.0) with SD as feeding medium (see subsection "Media preparation"). Different feeding strategies were tested. In DO-stat feeding fermentations, with DOC setpoint at 30%, the agitation rate was maintained at 800 rpm (after feeding initiation). In fed-batch fermentations with a linear ascending or pH-stat feeding, the DOC was maintained at 30% by cascading agitation (400–1000 rpm) with constant aeration rate (1 vvm).
For linear ascending feeding profile we used the following equation:
where F is the feeding rate (mL min−1), t the cultivation time after initiation of the fed-batch culture (min) and, a and b are constants for the linear ascending feeding profile .
Samples were withdrawn periodically for quantitative analysis along the cultivation. Cell growth was monitored by measuring the optical density at 600 nm (OD600 nm) in a spectrophotometer. One optical density unit was found to be equivalent to 0.3342 g L−1 of dry cell weight by gravimetric quantitation. Glucose concentration in the medium was measured with a glucose analyzer (model 2700 select, Yellow Springs Instruments, Yellow Springs, OH, USA). Acetate concentration was determined by high performance liquid chromatography (Äkta Purifier, GE HealthCare©, São Paulo, Brazil) equipped with an Aminex HPX-87H column (Bio-Rad Laboratories, Hercules, CA, USA), using 0.005 M H2SO4 as mobile phase and a UV-detector. The protein expression was analyzed by 12% SDS-PAGE stained with Coomassie® Brilliant Blue R-250 staining. The annexin V protein produced was quantified using the Qubit® Protein Assay Kit (Invitrogen™, Life Technologies, São Paulo, SP, Brazil) and a Qubit® 2.0 Fluorometer (Invitrogen™).
ANXA5 recombinant protein was purified using a Fast Performance Liquid Chromatography (FPLC) ÄKTA Purifier System (GE HealthCare©). All chromatographic steps were carried out at 4°C. Sample elution was monitored by UV detection at 215, 254 and 280 nm and fractions were analyzed by 12% SDS-PAGE. According to a previous report , frozen cells (3 g wet weight) were suspended in 30 mL of buffer A (50 mM Tris HCl, 10 mM CaCl2 pH 7.2) and incubated with 1 mM of phenylmethanesulfonylfluoride for 30 min at 4°C. The cells were disrupted by sonication (eight pulses of 10”) and centrifuged at 38,900 g for 30 min. The supernatant was discarded and the pellet was completely dissolved in 30 mL of buffer B (50 mM Tris HCl, 20 mM EDTA pH 7.2), stirred for 30 min at 4°C, and clarified by centrifugation at 38,900 g for 30 min at 4°C. The supernatant was dialyzed against 20 mM Tris HCl pH 8.0 (3 × 2 L, 3 h each). Residual precipitate was removed by centrifugation (38,900 g for 20 min) and the supernatant was loaded on a MonoQ HR 16/10 anion exchange column (GE Healthcare) previously equilibrated with 20 mM Tris HCl pH 8.0. Protein was eluted with 25% linear gradient of 20 mM Tris HCl, 1 M NaCl pH 8.0 at 1 mL min−1 flow rate. Homogeneous rhANXA5 was eluted at approximately 190 mM NaCl. Fractions containing homogeneous rhANXA5 were pooled, dialyzed against 20 mM N-2-hydroxyethylpiperazyne-N’-2-ethanesulfonic Acid (HEPES), 100 mM NaCl pH 7.2 and concentrated using an AMICON (Millipore Corporation, Bedford, MA, USA) ultra-filtration membrane (MWCO = 10 kDa), and stored at −80°C. Protein concentration was determined with Qubit® Protein Assay Kit using a Qubit® 2.0 Fluorometer.
rhANXA5 identification by mass spectrometry
rhANXA5 preparations (1 nmol) were desalted and subjected to proteolytic degradation using trypsin. The resulting peptides were separated by chromatography using 15 cm capillary columns (150 μm i.d., Kinetex C18 core-shell particles, Phenomenex, Inc., Torrance, CA, USA) and a nanoLC Ultra 1D plus equipment (Eksigent, Redwood City, CA, USA). Separated peptides were analyzed using an LTQ-Orbitrap hybrid mass spectrometer (Thermo Fisher Scientific Inc, Waltham, MA, USA). The chromatographic method used a step gradient from mobile phase A (0.1% formic acid in water) to mobile phase B (0.1% formic acid in acetonitrile): 0–2% B over 5 min; 2–10% B over 3 min; 10–60% B over 60 min; 60–80% B over 2 min; 80% B isocratic for 10 min; 80–2% B over 2 min; and 2% B isocratic for 8 min. We performed MS/MS fragmentation using collision-induced dissociation (CID) with an activation Q of 0.250, an activation time of 30.0 ms, and an isolation width of 1.0 Da. Using the Proteome Discoverer software (v. 1.3), we compared experimentally obtained MS and MS2 spectra with the in silico trypsin digestion of the human proteome. We allowed a precursor tolerance of 10 ppm, a fragment tolerance of 0.8 Da, static carbamidomethylation on cysteines, and oxidation on methionine residues. We restricted our analysis to matches with an Xcorr score > 2.0 for doubly charged ions and Xcorr score > 2.5 for triply charged ions.
Determination of rhANXA5 molecular mass
Purified rhANXA5 samples were desalted, reconstituted in acetonitrile 50%/MilliQ-water 49%/formic acid 1% and directly injected using a 500 μL syringe (Hamilton Company, Reno, NV, USA) in a static mode into an IonMax electrospray ion source. The electrospray source parameters were as follows: positive ion mode, 4.5 kV of applied voltage to the electrospray source, 5 arbitrary units (range 0–100) of sheath gas flow, 45.6 V of capillary voltage, 250°C of capillary temperature, and 238.8 V of tube lens voltage. Full spectra (600–2000 m/z range) were collected on a Thermo Orbitrap Discovery XL in profile mode using the linear ion trap analyzer (ITMS mode). The average spectrum was processed with the software MagTran  for charge state deconvolution.
FITC-annexin V binding test
To confirm the ability of rhANXA5 to detect cells undergoing apoptosis, we labeled rhANXA5 with the FluoroTag™ FITC Conjugation Kit (Sigma-Aldrich®). Our home-made kit also contained PI solution (Sigma-Aldrich®) and a 10 × binding buffer (0.1 M HEPES-NaOH pH 7.4, 1.4 M NaCl, 25 mM CaCl2). B16F10 melanoma cells (4X104), a kind gift from Dr. Peter Henson (National Jewish Center for Immunology, Denver, CO, USA) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), gentamicin 80 mg L−1 (Novafarma, Anápolis, Brazil), and Fungisone 5 mg L−1 (Bristol Myers Squibb, New York, NY, USA). To induce apoptosis, the cells were treated with cisplatin (Libbs, Embu, SP, Brazil) at different concentrations (0, 40, 80 and 160 μg/mL) and after 6 or 12 h, the cells were stained with our home-made kit or with a commercial kit from BD Biosciences (FITC Annexin Apoptosis Detection Kit II, BD Biosciences, San Diego, CA, USA). Cells were stained at a concentration of 105 cells mL−1 in 100 μL of 10X Binding Buffer using 5 μL of PI and 5 μL of rhANXA5 conjugated with FITC (home-made kit) or 5 μL of annexin-FITC from FITC Annexin Apoptosis Detection Kit II. All data were collected in a FACSCanto II flow cytometer (BD Bioscience) and analyzed using FlowJo software (Tree Stat, San Carlos, CA, USA).
Availability of supporting data
Supporting data are included in an additional file.