All restriction enzymes, DNA marker, and protein marker were purchased from Takara Biotechnology (Japan). The P. pastoris X-33, pPICZαC vector, and zeocin antibiotic were obtained from Invitrogen (CA, USA) and all primers were synthesized by Sangon Biotechnology Corp. (Shanghai, China). pPICZαC vector without cleavage of Ste13 was reconstructed by our lab. The murine anti-human urokinase monoclonal antibody was obtained from American Diagnostica Inc. (USA). PCR purification kit, gel extraction kit, and Miniprep kit for plasmid extraction were obtained from Sangon Biotechnology Corp. SP Sepharose XL and Source™30 RPC reversed phase hydrophobic chromatography were purchased from Phamarcia (Sweden). YPD, BMGY, BMMY media were prepared as formula (Invitrogen). HBL100 human breast epithelial cell line and SKOV3 human ovarian cancer cell line were obtained from the type culture collection of the Chinese Academy of Sciences (Shanghai, China).

P. pastoris was grown in YPD (2% peptone, 1% yeast extract, and 2% dextrose) or BMGY (0.1 M potassium phosphate, 2% peptone, 1% yeast extract, 1.34% YNB, and 1% glycerol). BMMY was used for protein induction (0.1 M potassium phosphate, 2% peptone, 1% yeast extract, 1.34% YNB and 0.5% methanol). YPD-zeocin plates (1% yeast extract, 2% peptone, 2% dextrose, 2% agar, and 0.1 zeocin) were used for selecting positive transformants.

The DNA sequence that encodes uPA amino-terminal fragment 1–43 amino acids and 26 amino acids of melittin was synthesized according to its native amino acid sequences and inserted into plasmid Bluescript II (pBs) by Sangon Biotechnology Corp. For the sake of insertion into pPICZαC vector and secreted expression the fusion protein, XhoI site and the Kex2 site were added to the 5′ end, termination codon and the EcoRI site were added to the 3′ end. In order to improve the yield of the fusion protein, synonymous codons were replaced by yeast biased codons. The full length gene which we designed to express rhuPA_1–43-melittin fusion protein was as followed: ctc gag aag aga tct aac gag ttg cac caa gtt cca tct aac tgt gac tgt ttg aac ggt ggt act tgt gtt tct aac aag tac ttc tct aac att cac tgg tgt aac tgt cca aag aag ttc ggt ggt caa cac tgt gag gtt att ggt gct gtt ttg aag gtt ttg act act ggt ttg cca gct ttg att tct tgg att aag aga aag aga caa caa tga gaa ttc.

The recombinant plasmid pBs-uPA_1–43-melittin was digested with XhoI and EcoRI, the resulting uPA_1–43-melittin DNA fragment then was inserted into the corresponding sites of the expression vector pPICZαC. Then the recombinant plasmid was transformed into competent cells of Escherichia coli XL-Blue and the recombinant colonies were selected by zeocin (25 μg/ml) resistance. Both the nucleotide sequences of the inserted DNA and flanking sequence were verified by sequencing with Genome Lab DTCS-Quick Start kit and CEQ 2000 DNA analysis system (Beckman, USA).

Transformation of P. pastoris and selection of high-level expression colonies. Plasmid DNA pPICZαC-uPA_1–43-melittin was linearized with SacI and introduced into Pichia host cells P. pastoris X33 by electroporation using a Micropulser (Bio-Rad, USA) according to the pPICZα vector manual. After the electroporation, 1 M ice-cold sorbitol was added immediately, and the cuvette contents were incubated at 30°C for 60 min. The mixture was spread on yeast extract peptone dextrose (YPD) agar plates containing zeocin and cultured at 30°C for two days. Antibiotic zeocin was used in the concentration of 0.1 g/l. After the transformants with zeocin resistance appeared, some monoclonal transformants were picked up randomly from the plates and initially cultured in a 50-ml conical tube containing 10 ml BMGY medium at 28°C with shaking at 225 rpm for 24 h. The cloned cells were then centrifuged and resuspended in 10 ml BMMY medium to induce expression for 7 days. The culture media (0.5 ml) were sampled per day and centrifuged at 4°C, 10,000 rpm for 5 min. Cell pellets and supernatant were separated. The supernatant was used for recombinant protein detection and the cell pellets were used for genomic DNA analysis. Methanol was added every 24 h to a final concentration of 0.5% (v/v) for inducing the expression of the target protein. The blank plasmids of pPICZαC were also transformed as a negative control.

In order to achieve high level expression of rhuPA_1–43-melittin, different culture parameters including pH value and induction time were evaluated in the expression procedure. The pH values were adjusted to 3.5–7.0 with 0.5 intervals. The processes were the same as above and the pH values were adjusted every day with disodium hydrogen phosphate and citric acid. At the desired time-points, 0.5-ml cell aliquots were withdrawn and then replaced with equal amount of fresh medium. The supernatant samples were used for enzyme-linked immunosorbent assay.

Individual wells of ELISA plate (Costar, USA) were coated with 50 μl supernatant sample of rhuPA_1–43-melittin which had been diluted with 50 μl coating buffer (15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.6) overnight at 4°C. The plates were blocked with 5% (w/v) non-fat milk powder in TPBS (PBS with 0.05% Tween-20) and incubated for 2 h at room temperature. The murine anti-human urokinase monoclonal antibody (American Diagnostica Inc., USA) was used at 1:1,000, incubated for 1 h at 37°C. Following several washes with TPBS, the plates were incubated with goat anti-mouse IgG conjugated to HRP (Dingguo, China) (1:250 dilution with blocking buffer) for 1 h again. The color reaction was developed by addition of the substrate solution ortho-phenylenediamine (OPD) and incubated for 5 min at room temperature in the dark. Then 50 μl stop solution (2 M H₂SO₄) was added to each well. The absorbance values at 490 nm were read in ELX800 Microplate Reader (Bio-Tek, USA). The reading work was finished within 2 h after adding the stop solution.

The highest-level expression transformant was cultured in a 5-l shake flask containing 2 l BMGY medium at 28°C until the culture reached OD600 = 6.0, the cells were harvested by centrifugation and resuspended in 2 l BMMY (pH 6.0) medium, and cultured at 28°C with shaking for 4 days. Sampling of the culture medium was performed every 24 h to analyze cell wet weight, optical density and determine rhuPA_1–43-melittin expression based on SDS-PAGE analysis, and the culture was supplemented daily with 10 ml methanol to a final concentration of 0.5% (v/v) for inducing the expression of rhuPA_1–43-melittin.

A cation exchange chromatographic column (20 ml, SP Sepharose XL, Sweden) was equilibrated with 100 ml 20 mM NaAc-HAc (pH 3.5) buffer. The supernatant was collected by centrifugation at 15,000 rpm for 10 min and was clarified with a 0.45-μm cellulose membrane. After being diluted four times with 20 mM NaAc-HAc (pH 3.5) buffer, the pH of the fermentation broth was adjusted to 3.5 with 1 M acetate acid. The supernatant was loaded onto the cation exchange chromatographic column at the rate of 0.5 ml/min. Then the column was washed extensively with the same buffer at the rate of 1 ml/min. The bound protein was eluted with a linear gradient of 0.1–1.0 M NaCl while the flow rate was maintained at the rate of 1 ml/min. Protein elution was monitored by measuring the absorbance at 280 nm and identified by SDS-PAGE analysis. Column effluent containing rhuPA_1–43-melittin was collected and submitted to Vivaflow 200 (Sartorius Stedim, Germany) at the rate of 1 ml/min to remove impurity proteins with molecular weight >10 kDa. Then the ultrafiltrate containing rhuPA_1–43-melittin was loaded onto a reverse phase column (2.0×15 cm, Source 30, Sweden) which was equilibrated with 0.1% trifluoroacetic acid (TFA) for further purification. RhuPA_1–43-melittin was eluted using 50% methanol containings 0.1% TFA and 100% methanol (containing 0.1% TFA) at the rate of 1 ml/min and monitored by measuring the UV absorbence at 280 nm. Column effluent containing rhuPA_1–43-melittin was concentrated by vacuum distillation and freeze drying to remove methanol. The finally purified rhuPA_1–43-melittin was stored at −80°C for further studies.

SDS-PAGE analysis was performed using an 18% gel. For western blotting, proteins in the gel were transferred to a nitrocellulose membrane. The membrane was blocked with 5% BSA for 1 h and then incubated with the murine anti-human urokinase monoclonal antibody for 12 h. After being washed, the membrane was incubated with the goat anti-mouse IgG conjugated to HRP (Dingguo), diluted to 1:250. The bound antibody was detected using 3,3′-diaminobenzidine (DAB).

To determine the N-terminal sequence, the purified rhuPA_1–43-melittin was electrophoresed on 18% SDS-PAGE gel and electroblotted on a PVDF membrane. After being blotted, the PVDF membrane was stained with Coomassie brilliant blue R250, and the rhuPA_1–43-melittin band was cut out and determined by automated Edman degradation performed on a model PPSQ-21A protein sequencer (Shimadzu, Japan).

The growth and proliferation of SKOV3 ovarian carcinoma cells line was observed with MTT assay. Briefly, human ovarian cancer SKOV3 cells were maintained in H-DMEM with 10% fetal bovine serum (FBS) and 100 U/ml of penicillin/streptomycin, at 37°C in humidified atmosphere containing 5% CO₂. Cells (1×10⁴) were seeded in 96-well plates containing complete medium and incubated for 24 h followed by different doses of rhuPA_1–43-melittin for 24 h. SKOV3 were treated by H-DMEM (as control) and 5, 10, 20, 40 and 80 μg/ml rhuPA_1–43-melittin, respectively. Then 20 μl MTT solution (5 mg/ml) was added to each well. After being incubated for a further 4 h at 37°C, the culture medium including MTT solution in the well was removed, and 150 μl DMSO was added to each well and mixed thoroughly to dissolve the crystals. Then the absorbance at 490 nm was detected (Bio-Rad Instruments, USA).

To test the effect of rhuPA_1–43-melittin on normal cell proliferation, human breast epithelial cells HBL100 were plated as above and incubated for 24 h followed by different doses of rhuPA_1–43-melittin for 24 h. HBL100 were treated by RPMI-1640 (as control) and 5, 10, 20, 40 and 80 μg/ml rhuPA_1–43-melittin, respectively. MTT assay was performed as above.

Cell cycle analysis was performed using propidium iodide staining. Briefly, the SKOV3 cells were seeded in a 6-well plate. After being treated by H-DMEM (as control) and 20, 40 and 80 μg/ml rhuPA_1–43-melittin, respectively, the cells were washed twice with phosphate-buffered saline (PBS), and then fixed in 70% ethanol overnight. After being washed twice in PBS, the cells were stained in propidium iodide (50 μg/ml) in the presence of 50 μg/ml RNase (DNase free) for 30 min. Flow cytometry (BD Biosciences, USA) was used to detect the cell cycle and a quantitative analysis of the cell cycle distribution was carried out using a trial version of ModFit LT V3.0 software (Verity Software House, USA). The results are expressed as percentage of the cells in each phase.

Apoptosis was determined by TUNEL assay. SKOV3 cells at density of 1×10⁵ cells/well were seeded onto 1.3 mm coverslips in 24-well plates and incubated for 24 h followed by different doses of rhuPA_1–43-melittin for 24 h. SKOV3 cells were treated by H-DMEM (as control), 20, 40 and 80 μg/ml rhuPA_1–43-melittin respectively and the cells were fixed with freshly prepared paraformaldehyde [4% in PBS (pH 7.4)], rinsed with PBS, and incubated in permeabilization solution. TUNEL staining was performed with an in situ cell death detection kit (Bioss, China) according to the manufacturer’s protocol. The TUNEL reaction mixture containing reaction buffer, terminal deoxynucleotidy1 transferase, and bromodeoxyuridine triphosphate was added onto the slides and incubated for 2 h at 37°C, followed by washing and incubation with an HRP-labeled anti-bromodeoxyuridine monoclonal antibody for 30 min at room temperature. The presence of antigen was then visualized with diaminobenzidine. Slides were subsequently counterstained with hematoxylin and imaged under bright-field microscopy. The number of TUNEL-positive cells was counted in six different fields and representative fields were photographed. The percentages of apoptotic cells were calculated from the ratio of apoptotic cells to total cells counted.

Results of DNA sequence analysis of the recombinant expression vector pPICZαC-uPA_1–43-melittin (data not shown) demonstrated that the DNA sequences encoding human uPA amino acids 1–43 and melittin were correctly inserted into pPICZαC vector and the amino acid sequence of uPA_1–43-melittin encoding was identical with that logged in GenBank.

After being cultured at 30°C for 2 days, dozens of transformants with zeocin resistance appeared on YPD agar plates which contained 0.1 g/l zeocin. The PCR analysis of genomic DNA showed that the DNA sequence encoding human uPA amino acids 1–43 and melittin was indeed integrated into over 90% of clones which transformed with recombinant expression vector pPICZαC-uPA_1–43-melittin. There were ~600 bp amplification bands for the samples that were transformed with pPICZαC-uPA_1–43-melittin, however, ~400 bp for the control sample which was transformed with pPICZαC blank plasmid (Fig. 1).

After induction with methanol for rhuPA_1–43-melittin expression, one of the transformants which presented the highest expression level of rhuPA_1–43-melittin could be used for further experiments. SDS-PAGE analysis of rhuPA_1–43-melittin culture medium indicated that rhuPA_1–43-melittin expressed after the induction of methanol, however, the rhuPA_1–43-melittin expression in the transformant containing blank plasmids of pPICZαC was negative. Based on the amino acid sequence, the calculated molecular weight of rhuPA_1–43-melittin was 7.6 kDa, which was consistent with the result of SDS-PAGE measurement (Fig. 2).

The transformant that presented the highest expression level was chosen for scaled-up protein production. After a series of experiments, the optimal expression conditions of rhuPA_1–43-melittin were obtained as follows: the optimal pH was 5.5 (Fig. 3a) and the optimal induction time-points was on the 4th day for the strain (Fig. 3b) at 28°C and with methanol daily addition concentration of 0.5% (v/v). Under these conditions, high level expression transformant of P. pastoris strain was obtained and retained for further studies.

The rhuPA_1–43-melittin supernatant was purified with a cation exchange chromatography and a reverse phase chromatography. Using an AKTA Explorer 100 chromatography system, we optimized the purification parameters. The optimal concentration of NaCl for elution was 0.6 M and 100% methanol (containing 0.1% TFA) can elute the bound rhuPA_1–43-melittin from the reverse phase chromatographic column (Fig. 4a). The concise purification protocol of rhuPA_1–43-melittin is presented in Table I.

The primary purified recombinant protein was identified by western blot analysis. The results demonstrated that the recombinant protein could bind with murine anti-human urokinase monoclonal antibody. No band was observed in lane 1, which is the supernatant before adding methanol (Fig. 4b).

N-terminal sequencing of rhuPA_1–43-melittin offered the first 14 amino acids as S N E L H Q V P S N C D C L. The N-terminal sequence of rhuPA_1–43-melittin was identical to that of human uPA, confirming the successful expression and purification of rhuPA_1–43-melittin.

After being treated with rhuPA_1–43-melittin at different doses, some SKOV3 cells showed membrane blebbing, ballooning and chromatin condensation. However, the cells grew well with adherence and the cells were fusiform or diamond shaped in the control group. MTT assay was used to detect the quantity of cells. As a result, rhuPA_1–43-melittin treatment brought about a dose-dependent inhibition of growth on SKOV3 at 24 h. The inhibition rate is ~84% at the dose of 80 μg/ml (Fig. 5). However, the result (Fig. 5) showed that rhuPA_1–43-melittin did not have much influence on the proliferation of normal cells (HBL100). The reason is that uPAR is highly expressed in ovarian cancer SKOV3 cells, but it is undetectable in normal cells such as HBL100. In other words, the anticancer effect of rhuPA_1–43-melittin can be exerted when uPA_1–43 combines with its receptor-uPAR followed by the release of melittin. So the fusion protein we expressed has no obvious toxicity on normal tissues and it can be applied to ovarian cancer targeted therapy.

To examine whether rhuPA_1–43-melittin that induced growth inhibition was associated with cell cycle regulation, the cell cycle distribution was analyzed by flow cytometry. After SKOV3 cells were incubated with rhuPA_1–43-melittin for 24 h, cells were harvested and further prepared for cell cycle analysis. Of the SKOV3 cells 41.95% was in the G1 phase under normal growth conditions, while treatment of SKOV3 cells with 20, 40 and 80 μg/ml rhuPA_1–43-melittin resulted in 47.85, 58.61 and 70.75% of cells in the G1 phase of the cell cycle, respectively (Fig. 6). These results indicate that rhuPA_1–43-melittin may induce G1 phase cell cycle arrest of SKOV3 cells.

The TUNEL assay demonstrated that ovarian cancer cells underwent apoptosis after treatment with rhuPA_1–43-melittin. Data from all designated fields were pooled to obtain the apoptotic index, which is the percentage of TUNEL positive cells in total cells manually counted in 6 random fields. The apoptosis index of rhuPA_1–43-melittin treatment groups were remarkably increased compared with the normal control group (P<0.001). As a result, rhuPA_1–43-melittin treatment caused a dose-dependent increase of apoptosis in SKOV3 within 24 h (Fig. 7).