Mice
Balb/c nude mice and all the mice used were purchased from the Shanghai Laboratory Animal Center, Chinese Academy of Sciences (Shanghai, China). All mice were housed and maintained in specific-pathogens-free conditions at the Institute of Health Sciences. All animal experiments were approved by the Institutional Biomedical Research Ethics Committee of the Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS) and performed in compliance with the Guide for the Care and Use of Laboratory Animals [14].
Strains and plasmids
The plasmid pPIC9K and the yeast cell strain GS115 were obtained from the Key Laboratory of Molecular Medicine of Fudan University [15]. The E. coli strain DH5α was obtained from TIANGEN Biotech Co., Ltd. (Beijing).
Yeast nitrogen base (with or without amino acids) was purchased from Sigma. Q-Sepharose-FF and Sephadex G-50 were purchased from GE Healthcare.
Construction of expression vector pPIC9K/RANK-N
The gene fragment encoding RANK-N (amino acids 29 to 213) is a part of the RANK gene (85–639 bp). A version of RANK-N gene fragment encoding RANK-N was codon optimized (Pichia pastoris), synthesized and cloned into a pUC57 plasmid (Additional file 1). The procedure was performed in Sangon Biotech. The RANK-N fragment was then integrated into pPIC9K that had been digested with XhoI and NotI (Thermo Scientific). The presence of pPIC9k/RANK-N in successful recombinant colonies was confirmed by gene sequencing.
Transformation and selection of recombinant P. pastoris colonies that highly express the target protein
The plasmid pPIC9K/RANK-N was cut with restriction enzyme SalI. The digested products were then purified using an EasyPure PCR Purification Kit (Transgene Biotech), electroporated with a MicroPulser™ Electroporator (Bio-Rad) and transformed into the yeast strain GS115. The transformants were cultured on MD plates (2% glucose, 4 × 10−5% biotin, and 1.34% YNB) for 2–3 days. We picked up about 1000 colonies for further screening with G418 (Amresco E859-5G). Strains that could grow at high concentrations (2 mg/ml or more) of G418 were stored at −80 °C.
Each clone obtained was then streaked on a new YPD plate to obtain single colonies. Next step, each single colony was inoculated in 250 mL flasks with 50 ml BMGY medium and cultured at 30 °C in a shaker at 220 rpm. When the OD600 reached 3–4 (logarithmic phase), we harvested and rinsed the cells. Then rinsed cells were resuspended in 50 mL BMMY and cultured in the same shaker of 220 rpm at 30 °C, and the culture was supplemented with 500 μl methanol to a final concentration of 1% every 12 h. An 80-μl aliquot of the supernatant was collected at every 12 h so that the expression of RANK-N could be determined using SDS-PAGE and western blotting.
Purification of the RANK-N
After 96 h of induction, the total supernatant from all the flasks (approximately 2 l) was applied into an ultrafiltration system (Merck Millipore, P2PLBCC05) and concentrated to approximately 200 mL in volume. Then, the 200-mL sample was loaded to a Sephadex G-50 size exclusion column which had been preequilibrated with Tris-HCl buffer (20 mM, pH 9.0). The fraction with RANK-N was then applied to a Q-Sepharose-FF column installed in an ÄKTA explorer 100 system at 5 mL/min. The column was washed with Tris-HCl buffer (20 mM, pH 9.0) until the absorbance at 280 nm reached the base level. After that, a linear gradient of washing buffer (1.0 mol/L NaCl in 20 mM Tris-HCl pH 9.0) was pumped to the column to wash the protein off from the column. The protein fractions with RANK-N was harvested, lyophilized and stored at −80 °C.
Western blot and Coomassie Brilliant Blue staining
SDS-PAGE analysis was carried out according to the standard protocol. Gels were stained in Coomassie Brilliant Blue R-250 solution overnight or for at least 3 h and then destained in destaining buffer. For WB analysis, the proteins in the gel were transferred to a PVDF membrane (Immobilon P, Millipore) using a wet electroblotting system (Bio-Rad) working at 100 V for 50 min in a Tris-glycine running buffer (192 mM glycine and 25 mM Tris). The membrane with protein was then blocked in 5% non-fat milk for 1 h at room temperature (RT), after which it was first immunoblotted with specific antibodies at 4 °C overnight and then with HRP-conjugated secondary antibody for 1 h at Room Temperature. The signals of the bound antibodies were detected using Super Signal West Pico Chemiluminescent Substrate (Pierce) in a LAS4000 camera system (Fujifilm). The specific antibody against RANK-N (sc-7626) was purchased from Santa Cruz Biotechnology.
Biacore T100 analysis
Surface plasmon resonance experiments were carried out with a Biacore T100 protein interaction analysis system (GE Healthcare, Little Chalfont, UK). RANKL (R&D Systems, 462-TEC-010/CF) protein was covalently coupled to a CM5 sensor chip (GE Healthcare) using an amine coupling kit (GE Healthcare). RANK-N was diluted with PBS-EP + running buffer (3 mM EDTA, 150 mM NaCl, 10 mM HEPES and 0.05% (v/v) Surfactant P20) and then injected over the sensor chip at 30 μl min−1 at 25 °C (contact time: 210 s, dissociation time: 400 s). After each measurement of a binding response (in resonance units, RU), the sensor chip was regenerated using 10 mM glycine–HCl buffer (pH 2.5). The values of dissociation rate constant (k
d
), the association rate constant (k
a
) and equilibrium dissociation constant (K
D
) were derived by simulating the binding curves with a monovalent interaction model using BIA evaluation software (GE Healthcare).
Sugar content analysis of the recombinant protein
A total of 10 μg of purified RANK-N was treated with 1 μL PNGase F (NEB, P0704S) and 2 μL of 10% NP40 for 1 h, and then denatured at 100 °C for 10 min according to the manufacturer’s instruction. Finally, the treated protein was analyzed using Coomassie Brilliant Blue staining.
Inhibition of bone marrow-derived macrophage differentiation into osteoclasts by RANK-N
Bone marrow-derived macrophages were cultured in RPMI-1640 medium containing 10% (vol/vol) FCS, 50 μM 2-mercaptoethanol and 10 ng/ml M-CSF (PeproTech). Then, 1 μg/ml RANKL was added to induce bone marrow-derived macrophages (BMDMs) to differentiate into osteoclasts, and TRAP staining (Sigma) was performed to evaluate the differentiation process.
PDX tumor model experiment
Colorectal tumors were collected at the time of surgery, washed with phosphate-buffered saline (PBS) and injected subcutaneously into the flanks of 6-week-old athymic mice after being cut into pieces approximately 2 mm in size. Mice were observed daily, and tumors were measured with vernier calipers until the volume of the tumor (V = L × 2 W × 0.52 (L = longest diameter, W = shortest diameter)) reached approximately 1000 mm3. Tumors were then harvested, minced and reimplanted as described above until stable PDX was established.
The tumor-grafted mice were blindly divided into two groups (4 mice per group), and RANK-N (100 mg/kg) or PBS was injected intraperitoneally (i.p.) every other day for a total of 2 months. Antitumor activity of treatments was evaluated by tumor weight and tumor volume. Protein was extracted from the tumor samples, and further analyzed using western blotting.
Data analysis and statistics
Statistical analysis was carried out using GraphPad Prism 5.0 software. The data are presented as the mean ± SD and analyzed using unpaired T. test, p < 0.05 was considered statistically significant. All experiments in this study were performed at least three times.