Expression and purification of bomapin from E. coli
The BamH1 and Xho1 restriction sites in the pET15b vector (Novagen) were flipped using Quick Change Site-Directed Mutagenesis Kit (Stratagene) and primers: 5'-CTGGTGCCGCGCGGCAGCGGATCCCTCCTCGAGCCGGCTGCTAACAAAGCCCG-3' and 5'-CGGGCTTTGTTAGCAGCCGGCTCGAGGAGGGATCCGCTGCCGCGCGGCACCAG-3'. Bomapin cDNA was excised from the PGEX-4T-1-bomapin vector (a kind gift from Dr. R Schleef ) using Xho1 and BamH1, and cloned to the "flipped" pET15b vector. The C395S mutation was introduced with primers: 5'-CTTTTTTATGGAAGATTATCCTCC CCCTAA-3' and 5'-TTAGGGGGAGGATAATCTTCCATAAAAAAG-3', and followed by full-length cDNA sequencing. E. coli AD494(DE3) (Novagen) was then transformed and induced with 0.1 mM IPTG (isopropyl-β-D-thiogalactoside) to produce histidine-tagged bomapin. Bomapin was purified under native conditions on a TALON column (Clontech Laboratories) according to the manufacturer's instructions. Then, it was desalted on a NAP-25 column, loaded onto a MonoQ HR 5/5 column (both GE Healthcare), and eluted with NaCl gradient in 20 mM HEPES, pH 7.0. The yields of purification of wt bomapin and the C395S mutant were 1.2 mg and 0.13 mg per 1 L of bacterial culture, respectively.
Assay for inhibitory activity of bomapin
The inhibitory activity of bomapin against bovine trypsin (Sigma) was measured by a chromogenic assay using the substrate S-2488 (Chromogenics). Bomapin and trypsin were diluted in activity assay buffer (0.05 M Tris/HCl, pH 7.5, containing 0.15 M NaCl and 0.05% Tween 80) to final concentrations 15 μg/ml and 10 μg/ml, respectively. Trypsin (10 μl) was mixed in a 96-well plate with various amounts of bomapin to obtain bomapin/trypsin molar ratios 0.87, 1.74, and 4.3 (in total volume 100 μl), and incubated for 5 min at room temperature. Then 100 μl of 0.4 mM S-2488 substrate was added and absorbance at 405 nm was measured at 1 min intervals in a Titertek Multiscan spectrophotometer.
We have used leukaemia cell lines originating from different subclasses of myeloid leukaemia. However, the common characteristic of these cells is that their differentiation is blocked at early stages of myeloid differentiation: the HL-60 (acute promyelocytic leukaemia) and K562 (chronic myelogenous leukaemia) cells represent multipotent myeloid progenitors, and U937 (histiocytic lymphoma) and THP-1 (acute monocytic leukaemia) cells represent monocytic progenitors. The K562 cells (naturally bomapin-deficient), as well as U937, HL60, and THP1 cells (all expressing bomapin) were cultured in RPMI-1640 medium. The human fibrosarcoma HT-1080 cells (bomapin-deficient) were cultured in DMEM medium. All media were substituted with 10% foetal bovine serum (FBS), 2 mM L-glutamine, streptomycin (100 μg/ml), and penicillin (100 U/ml) (all from Gibco BRL). All the cells were from ECACC.
Expression of bomapin in eukaryotic cells and proliferation assays
Bomapin cDNA was amplified from the PGEX-4T-1-bomapin vector by PCR using pfu-DNA polymerase and primers 5'-AAAGCTGAATTCTCGAGGCACCATGGACTCTCTAGCAACATCAATC-3' and 5'-GGCGACCGGATCCGCGGGGGAGCATAATCTTCCATAAAA-3' containing XhoI and BamH1 cleavage sites (respectively). The PCR product was cloned into pd2EGFP-N1 vector (Clontech) using the above restriction sites, and fully sequenced. The C395S mutation was introduced with primers: 5'-CTTTTTTATGGAAGATTATCCTCCCCCTAA-3' and 5'-TTAGGGGGAGGATAATCTTCCATAAAA AAG-3', and followed by full-length cDNA sequencing. The pd2EGFP-N1 vector encodes for a destabilized bomapin-EGFP fusion protein with a half-life of about 2 h. K562 cells were transfected using lipofectamine 2000 (Life Technologies), selected with Geneticin (0.6 mg/ml, Life Technologies), and sorted with a fluorescence-activated cell sorter (Becton Dickinson). To correct for clone variation, all experiments were performed on a multiclonal pool of the stably transfected cells. Proliferation of K562 cells expressing wt bomapin was increasing with increasing cell generation number, whereas proliferation of K562 expressing the C395S bomapin mutant was decreasing with higher generation number. Cell proliferation was assayed by manual counting of trypan blue-excluded cells, and with Cell Proliferation Reagent WST-1 (4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate; Roche).
Apoptosis in K562 cells and the K562 cells stably expressing EGFP or bomapin-EGFP was induced by serum starvation. At different time points, the amount of dead cells was quantified by manual cell counting in the presence of trypan blue. For detection of apoptotic cells, 1 × 105 cells in 20 mM Hepes pH 7.1, supplemented with 0.14 M NaCl and 25 mM CaCl2, were incubated with annexin V-PE-Cys5 (Abcam) according to manufacturer's instruction, and cell-surface annexin-labelled cells were counted under fluorescent microscope (with excitation and emission wavelengths 488 nm and 670 nm, respectively); non-labelled cells were counted under normal phase light. In addition, cell extracts at different times of serum starvation were prepared in 10 mM Tris/HCl, pH 7.5 with 0.1 M NaCl, 1 mM EDTA 1% Triton X-100, and protease inhibitor cocktail (Roche Diagnostic), and analysed by western blot with antibodies for two apoptotic markers: lamins-A/C (rabbit antibodies against C-terminal fragment of Lamin A; Biolegend) and cleaved PARP (rabbit antibodies specific for cleaved PARP; Abcam), and for β-actin (with monoclonal antibodies to human β-actin; Sigma) as loading control.
For cell-cycle analyses, cells were fixed in 70% ethanol, treated with RNase A (500 μg/ml), and stained with propidium iodide (10 μg/ml). Cell cycle analysis was performed on a flow cytometer (Cytomix FC500; Backman Coulter).
Antibodies against bomapin and ELISA
His-tagged bomapin was used for immunization of a rabbit and chicken. Total IgY were purified from egg yolk (all at AgriSera AB, Sweden). This IgY and rabbit antiserum were depleted from anti-his-tag antibodies on immobilized his-tagged PAI-2 (serpinb2), and bomapin-specific antibodies were purified on bomapin-NHS-Sepharose. These antibodies were used for immunoprecipitation, western blot, and ELISA. For ELISA, IgY (2 μg/ml) were used for coating, and rabbit anti-bomapin antibodies (0.2 μg/ml) as first antibodies; these were followed by anti-rabbit horseradish peroxidase-conjugated secondary antibodies (Promega). The working range for the ELISA was from 1 to 60 ng/ml. For immunostaining, the antibodies were further pre-incubated with proteins from K562 cell extract that were immobilized on NHS-Sepharose.
The cells (2-5 × 108 cells) were lysed in 50 mM HEPES pH 7.4, 150 mM NaCl, 5% glycerol, 2.5 mM EDTA, 1% NP-40 and protease inhibitor cocktail (Roche Diagnostic). The cell lysates were centrifuged, and then immunoprecipitated with anti-bomapin IgY immobilized on NHS-Sepharose. The samples were analyzed by SDS-PAGE followed by western blot. Bomapin was detected using rabbit anti-bomapin antibodies, followed by anti-rabbit horseradish peroxidase-conjugated secondary antibodies (Promega). Immunoreactive bands were visualized with the Enhanced Chemiluminescence Kit (ECL, GE Helthcare).
Immunofluorescence microscopy of cells
Cells were seeded on poly-lysine coated cover slips (Sigma), fixed in 3% paraformaldehyde, permeabilised with 0.1% Triton X-100, and blocked with 2% goat serum in PBS. Bomapin was stained with rabbit anti-bomapin antibodies, followed by appropriate secondary antibodies conjugated with Alexa Fluor 568 (Molecular Probes). In the case of cells expressing bomapin-EGFP-fusion and EGFP, fluorescence of EGFP was detected. Images were captured with oil immersion using an AxioImager.Z1 microscope with ApoTome, lenses PLAN-APOCHROMAT 63 × 1.4, Axio Cam MRm (all from Zeiss) and Axio Vision software version 4.5. For quantification of the nuclear size, proliferating K562 cells expressing EGFP or bomapin-EGFP were DAPI-stained, the images were captured, and the nuclear areas were measured using the Axio Vision software.
U937 cells were incubated with antisense (BAS3: 5'-GCCCTATACTTTAAAGGAATC-3') or sense (BS: 5'-GCCCTATACTTTAAAGGAATC-3') phosphorothioated DNA oligonucleotides (DNA Technology A/S, Denmark) at 20 nmol/ml, using standard growth conditions. Cell proliferation was quantified by manual counting and with Cell Proliferation Reagent WST-1. Bomapin expression was assessed by immunoprecipitation from equal amounts of cell extracts (0.7 mg total protein) using anti-bomapin IgY immobilized on NHS-Sepharose. This was followed by western blot with bomapin-specific rabbit antibodies. For loading control, a small amount of IgY that normally detaches from IgY-Sepharose during the immunoprecipitation was detected with rabbit anti-chicken antibodies coupled to alkaline peroxidase (Sigma).
In silico models of reduced and oxidized bomapin
The model of reduced bomapin was generated from the bomapin sequence (P48595) using the SWISS-MODEL server (version 3.5)  with the coordinates of PAI-2 (1by7), serpinB1 (1hle), and ovalbumin (1ova) as templates. This model was energy-minimized further and used to calculate the oxidized bomapin structure using parallhdg.pro force field within X-PLOR version 3.851 . To create the C68-C395 disulfide bond, residues between positions 60-87 and 394-397 were allowed to move freely under the simulated annealing  and simulated annealing refinement protocols, whereas all other residues were kept in fixed positions to preserve the secondary structure elements and the overall fold of bomapin. The disulfide bond was introduced as a distance constraint (2.02 ± 0.05 Å) between the two sulphur atoms of the cysteines. Altogether, 20 structures were calculated and the structure with the lowest total energy was chosen as being representative of the ensemble of structures. The models of reduced and oxidized bomapin have been deposited to the Protein Model Data Base http://mi.caspur.it/PMDB/ with accession codes PM0074678 and PM0074679, respectively.
SDS-PAGE (10%) was performed as described previously , and followed by Coomassie Blue staining or immunoblotting . Protein concentration was determined using the bicinchoninic acid assay (Pierce) or the Coomassie Plus Protein Reagent (Bio-Rad), with bovine serum albumin as protein standard.
Data are expressed as mean values ± SEM, and analysed for statistical significance using uncoupled t-student test with confidence 95%; p value of <0.05 was considered as significant.