Expression of rBMP-2 in E. coli
The mature human BMP-2 coding sequence was purchased from the American Type Culture Collection (ATCC, Rockville, MD) as a lambda gt10 phage and amplified by the polymerase chain reaction (PCR) using the following primers:
5′ primer: 5′
3′ primer: 5′
NcoI and BamHI restriction endonuclease recognition sites are shown underlined within the sequences. A start codon (ATG) was contained within the NcoI site and a stop codon (TAA) was introduced immediately prior to the BamHI site. The NcoI and BamHI digested fragment was cloned into pET-15b (Novagen, San Diego, CA) as shown in , and the coding sequence verified by DNA sequencing.
Expression, Purification, and Bioactivity of Recombinant BMP-2
Expression vectors were transformed into competent cells BL21(DE3) bacteria and overnight cultures of single colonies were inoculated with 5 ml of Luria-Bertani broth supplemented with 100 μg/ml ampicillin and grown at 37°C for 4.5 h. Cultures were then induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 4 h. Induced cultures were centrifuged at 5000 × g for 20 min and the pellets were suspended in sample buffer or stored at −20°C. Lysates and purified proteins were separated on 4 to 20% gradient gels and stained with Coomassie Brilliant Blue R250 (EMD Chemicals, Gibbstown, NJ). The colony with the highest level of rBMP-2 expression was chosen for subsequent purification.
Purification and Refolding of rBMP-2
Purification and refolding were performed as described previously [17
] with slight modifications. Briefly, cells were induced as described above, then the cell pellet was washed twice with 50 mM sodium phosphate buffer (pH 7.0) and frozen at −80°C until use. The frozen pellet was resuspended in 20 mM Tris-HCl (pH 8.5), 0.5 mM EDTA, and 2% (v/v) Triton X-100. After vigorous vortexing, the suspension was ultrasonicated for 2 min on ice and centrifuged at 26,000 × g for 30 min at 4°C. This last step was then repeated to yield washed inclusion bodies.
The washed inclusion bodies were solubilized with 6 M guanidinium hydrochloride (Gnd-HCl) and soluble rBMP-2 was refolded for at least 72 h at RT in the presence of 6 M Gnd-HCl, 0.75 M 2-(Cyclohexylamino)ethanesulfonic acid (CHES) buffer, pH 8.5 and 3 mM glutathione (2:1 ratio of reduced:oxidized) with constant stirring. The renaturation mixture was extensively dialyzed against 4 M urea in 20 mM Tris-HCl (pH 8.0) and passed through a 0.22 μm filter (Minisart, Sartorious, Göttingen, Germany). 10 mg of total protein was applied to a 5-ml HiTrap heparin-sepharose column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and equilibrated with 5 column volumes (CV) of dialysis buffer consisting of 4 M urea and 20 mM Tris, pH 8.0. The column was washed again with 5 CV of dialysis buffer, and the monomeric and dimeric forms of rBMP-2 were eluted using 350 mM and 500 mM of NaCl, respectively. The eluted dimer fraction was concentrated to 1 mg/ml, and urea was removed, by repetitive ultrafiltration using a 5,000 MWCO spin cartridge (Vivaspin, Vivascience, Germany) and 50 mM 2-(N-Morpholino)ethanesulfonic acid (MES) buffer, pH 5.0. For long-term storage, rBMP-2 was freeze-dried in 50 mM MES (pH 5.0) without loss of biological activity.
Measurement of rBMP-2 Biological Activity
C2C12 cells from the ATCC were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS) and 1% L-glutamine at 37°C in a humidified atmosphere with 5% CO2
. The alkaline phosphatase (ALP) activity assay has been described in detail previously[18
]. Briefly, 3 × 104
cells per well were plated in 96-well plates (Nunc, Roskilde, Denmark) and cultured in 2% FBS, 1% L-glutamine and rBMP-2 at the concentration specified. rBMP-2 from a commercial source (R&D Systems, Minneapolis, MN) was used as a positive control. After 72 h, cells were washed with phosphate buffered saline (PBS), pH 7.4 and alkaline phosphatase activity of cell lysates was measured on a Versamax (Molecular Devices, Sunnyvale, CA) absorbance plate reader using pNPP (Sigma-Aldrich) as the substrate.
Radiolabeling of rBMP-2
The N-hydroxysuccinimide (NHS) ester of [99m
] was prepared with high radiochemical purity (>99%) and high specific activity (≈ 6,675 Ci/mmol) in dimethyl sulfoxide (DMSO) using solid-phase pre-loading technique within 20 min as described in detail previously [19
]. For radiolabeling, 1 nmol (12.5 μg) of rBMP-2 as a dried powder was resuspended in 50 μL of DMSO. Of note, rBMP-2 resuspended in DMSO retained its bioactivity after dilution into aqueous buffer (data not shown). Conjugation was performed by the addition of 500 μL of [99m
]-NHS (4 mCi, 0.36 nmol) to rBMP-2 in DMSO, followed by 20 μmol of triethylamine from a 4 M stock, and constant stirring at RT for 1 h. [99m
]-rBMP-2 was purified to homogeneity by gel-filtration chromatography using a 8 × 300 mm, 60 Å Diol (Catalog #DL06S053008WT; YMC, Kyoto, Japan) gel-filtration column and PBS, pH 7.4 as mobile phase. Purified [99m
]-rBMP-2 was concentrated to 1 mCi/ml using a 3,000 MWCO Vivaspin cartridge prior to injection.
Syngeneic Rat Model of Breast Cancer Microcalcification
Animals were used under the supervision of an approved institutional protocol. Female Fischer 344 rats were from Taconic Farms (Hudson, NY). At the time of injection, rats averaged 7 to 9 weeks of age and weighed 130 g ± 20 g. For tumor cell inoculation, rats were anesthetized with 2% isoflurane/balance O2
. R3230 cells adapted to cell culture [15
] were grown in DMEM supplemented with 10% FBS and antibiotics (penicillin [100 U/ml] and streptomycin [100 mg/ml]). After trypsinization, ≈ 2 × 107
cells in 0.3 mL DMEM were injected subcutaneously into the fat pad. Bioactive rBMP-2 (10 μg, 50 μg, and 100 μg) or vehicle control (PBS) was administered as a single intraperitoneal (IP) injection into R3230 tumor-bearing rats (n = 4 animals per group) at 4 days post-tumor cell inoculation. Resected tumors were fixed in formalin, processed for histology, and stained with hematoxylin and eosin (H&E) or by the method of von Kossa [20
Biodistribution and Clearance of [99mTc-MAS3]-rBMP-2
100 μCi [99mTc-MAS3]-rBMP-2 was injected IV or IP into groups of four R3230-bearing Fischer 344 rats. Blood samples were collected at 0 min, 5 min, 15 min, 30 min, 1 h, 2 h, 3 h, and 4 h. Rats were sacrificed at 4 h post-injection, total body radioactivity was measured in a dose calibrator (Capintec, Ramsey, NJ), then tumor, organs, and tissues were dissected, rinsed briefly with saline, and weighed. Radioactivity was quantified using a model 1470 Wallac Wizard (Perkin Elmer, Wellesley, MA) ten-detector gamma counter.
Multi-Modality Imaging of Breast Cancer Microcalcification
Tumor growth and tumor calcification were assessed weekly using micro SPECT/CT. 1 mCi of 99mTc-methylene diphosphonate (99mTc-MDP) in 0.2 ml saline was injected intravenously 4 h before imaging. Animals were anesthetized with 2% isoflurane/balance O2. Imaging was performed on a NanoSPECT/CT (Bioscan, Washington, DC) scanner equipped with an 8W x-ray source running at 65 kV (123 mA), and a 48 μm pitch CMOS-CCD x-ray detector. Continuous helical micro CT scanning was employed with the following parameters: 1 sec exposure, 240 angles, 1.3 magnification, 37 mm pitch (1 field-of-view), and a 512 × 256 pixel frame size (192 μm pixels). Images were reconstructed as 170 × 170 pixel transverse matrices with varying axial length and slice thickness of 0.4 mm (isotropic voxel size 0.4 mm) using filtered-back projection (SheppLogan filtering). Helical micro SPECT was performed using a four-headed gamma camera outfitted with multi-pinhole collimators having 2.5 mm diameter pinholes (36 total). Images were acquired over 360° in 48 projections of 50 sec each using a 256 × 256 frame size (1.0 mm pixels). The micro-SPECT images were reconstructed as 86 × 86 pixel transverse matrices with varying axial length and slice thickness of 0.8 mm (isotropic voxel size 0.8 mm). Quantitation of tumor volume, percent calcification, and 99mTc-MDP update was performed using InVivoScope software (Bioscan).
All experiments were repeated at least twice. A Student’s t-test was used to examine the differences between the experimental groups.