E. coli DH5α (Shanghai Sangon Biological Engineering Co. Ltd., Shanghai, China) was used for plasmid amplification and P. pastoris X-33 was used for the expression of the fusion protein. The pPICZα-A expression vector and P. pastoris X-33 were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). The restriction endonuclease, T4 DNA ligase and Taq DNA polymerase were purchased from Takara Biotechnology Co., Ltd. (Dalian, China). Ni-chelating Sepharose columns were purchased from Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China).

The HepG2 cell line was obtained from the Medical College of Henan University of Science and Technology (Luoyang, China). The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Shanghai Sangon Biological Engineering Co. Ltd.) supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin in a humidified 5% CO₂ atmosphere at 37°C.

Based on the primary amino acid sequences of the mature peptide and according to codon preference of P. pastoris X-33, the gene sequence encoding PG-1 (Fig. 1A) was synthesized and used to construct the plasmid pUC57-PG-1 (Shanghai Sangon Biological Engineering Co. Ltd.). An XhoI restriction enzyme site was introduced to allow in-frame cloning into the α-factor secretion signal of the pPICZα-PG-1 shuttle vector. A sequence encoding the Kex2 cleavage site (LEKR) and a 6His-tag were added upstream and downstream of the PG-1 codon sequence, respectively. A termination codon of PG-1 was introduced at the C-terminus, along with a XbaI restriction enzyme site (Fig. 1B).

With the plasmid pUC57-PG-1 as the template, the gene encoding PG-1 was amplified by polymerase chain reaction (PCR) with the common primers pUC57+ (5′-ATCAGGCGCCATTCGCCATTC-3′) and pUC57- (5′-CAGGTTCCCGACTGGAAAG-3′). The PCR mixture had a total volume of 50 μl and comprised 5 μl 10X PCR buffer, 4 μl dNTP mixture, 1 μl 20 μl/ml primer pUC57+, 1 μl 20 μl/ml primer pUC57-, 2 μl template, 1 μl Taq DNA polymerase and 36 μl ddH₂O. The PCR procedure involved 4 min predenaturation at 95°C; 30 sec denaturation at 94°C, 30 sec annealing at 53°C and 30 sec extension at 72°C for 30 cycles; followed by 3 min extension at 72°C. The amplified products were verified by agarose gel electrophoresis. The PCR products were and the pPICZα A plasmid were double digested with XhoI and XbaI respectively, and ligated with T4 DNA ligase to generate the fusion vector pPICZα-A. The plasmid was then digested with the same restriction enzymes, to generate the fusion vector pPICZα-A-PG-1. The ligation mixture was transformed into E. coli DH5α, and recombinant E. coli cells were selected on Zeocin-containing lysogeny broth plates (Invitrogen Life Technologies). The recombinant plasmid pPICZα-A-PG-1 was confirmed by restriction endonuclease digestion and DNA sequencing analysis.

pPICZα-A-PG-1 was linearized with SacI and transformed into competent cells of P. pastoris X-33 (Mut⁺) by electroporation (Gene Pulser Xcell™ Electroporation System; Bio-Rad, Hercules, CA, USA), according to the manufacturer’s instructions (Pichia Expression kit; Invitrogen Life Technologies). The empty pPICZα-A vector was similarly linearized and transformed into P. pastoris X-33 as a negative control. All Zeocin-resistant colonies growing in Yeast Extract Peptone Dextrose medium (1% yeast extract, 2% peptone, 2% dextrose, 1 M sorbitol, 2% agar and 100 mg/ml Zeocin) plates were plated in duplicate onto either minimal methanol with histidine [MMH; 1.34% yeast nitrogen base (YNB), 0.05% biotin, 0.5% methanol and 1.5% agar] or minimal dextrose with histidine (MDH; 1.34% YNB, 0.05% biotin, 1% dextrose and 1.5% agar) plates to characterize the methanol-utilizing phenotype. Mut⁺ strains were obtained from the MDH plates and the inserts were evaluated by PCR amplification of the yeast genomic DNA template, using the common aldehyde oxidase 1 primers (5′-GACTGGTTCCAATTGACAAGC and 5′-GCAAATGGCATTCTGACATCC). These strains were subsequently used for the suspension culture.

The positive P. pastoris transformants were selected and inoculated in 10 ml buffered glycerol-complex medium (1% yeast extract, 2% peptone, 100 mM potassium phosphate buffer pH 6.0, 1.34% YNB, 4×10⁻⁵% biotin and 1% glycerol) for 24 h at 28°C. When the cell density reached ~0.6 at optical density (OD)600 (725 UV Visible Spectrophotometer; Shanghai Precision & Scientific Instrument Co., Ltd., Shanghai, China), the cells were harvested by centrifugation at 3,000 × g for 2 min and resuspended to an OD600 of 1.0 in buffered methanol-complex medium (1% yeast extract, 2% peptone, 100 mM potassium phosphate buffer pH 6.0, 1.34% YNB, 4×10⁻⁵% biotin and 1.0% methanol). The cells were then cultured for 4–5 days at 28°C in a flask while adding methanol to a final concentration of 1% every 24 h. The cell culture medium was harvested by centrifugation at 12,000 × g for 20 min. The presence of the fusion protein in the supernatant was analyzed by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE) with Coomassie Brilliant Blue staining (16). As shown in Fig. 2, a major protein recombinant PG-1 band of ~2.9 kDa appeared following induction for 4 days. The protein concentration was determined using Bio-Rad Protein Assay Dye Reagent with bovine serum albumin (BSA) as standard (17).

The expression supernatant was applied to a Ni-chelating Sepharose column (1.6×10 cm) pre-equilibrated with phosphate-buffered saline to purify the PG-1 fusion protein. Subsequently, the column was washed with 20 bed volumes of the same buffer to remove contaminating proteins. The protein was eluted with a linear gradient from 0.1 to 1.0 M imidazole in 1X Ni-NTA elution buffer (50 mM NaH₂PO₄ pH 8.0 and 300 mM NaCl). The peak amount of PG-1 eluted was determined by analysis of the fractions with 15% Tricine-SDS-PAGE, and the fractions containing PG-1 were collected. The collected fractions were dialyzed in Milli-Q water (Millipore, Billerica, MA, USA). The protein concentration of the purified PG-1 was determined using Bio-Rad Protein Assay Dye Reagent with BSA as standard. Subsequently, the purified peptide was lyophilized and stored at −20°C until use.

The cytotoxicity of the purified PG-1 fusion protein was determined by a tetrazolium (MTT) assay (18). The HepG2 cells (3×10³ cells/well) were plated in 100 μl DMEM per well in 96-well plates (Costar Corning, Corning, NY, USA). Following overnight incubation, purified PG-1 was added in various concentrations (10, 20, 40, 60 and 160 μg/ml) to the wells, with six wells for each concentration. After treatment with purified PG-1 for 1, 2, 3, 4 and 5 days, 20 μl 5 mg/ml MTT (pH 4.7) was added to each well and the cells were cultivated for a further 4 h. The supernatant fluid was then removed, 100 μl DMSO was added to each well and the samples were agitated for 15 min. The absorbance at 490 nm was measured with a microplate reader (Bio-Rad) using wells without cells as blanks. Three independent experiments were performed for each set of conditions.

Using degenerate primers, the PG-1 gene was amplified by PCR and verified by agarose gel electrophoresis. The PCR product was ligated into the pPICZα-A expression vector together with an α-mating factor secretion signal sequence at the N-terminus and a 6His-tag at the C-terminus of the PG-1 peptide. The recombinant plasmid pPICZα-A-PG-1 was successfully constructed and verified by restriction enzyme analysis and DNA sequencing.

The recombinant plasmid pPICZα-A-PG-1 was linearized with SacI and transformed into competent cells of P. pastoris X-33. The pPICZα-A-PG-1 transformants of P. pastoris were grown in flasks at 28°C and, after culture for 120 h, the cell culture medium was harvested by centrifugation. The supernatant from the flask culture was analyzed by Tricine-SDS-PAGE. As shown in Fig. 2, a major band at ~2.9 kDa was observed after a 96-h induction. The protein concentration in the supernatent was 15.6mg/100ml, as measured using Bio-Rad Protein Assay Dye Reagent Using an Ni-NTA column, the fusion protein was eluted at 250 mM imidazole. The eluted fractions were collected and dialyzed, and the results of the Tricine-SDS-PAGE (Fig. 3) indicated that the recombinant PG-1 fusion protein (2.9 kDa) had been obtained with high-purity. Approximately 20 mg PG-1/6His was purified from 500 ml culture medium.

The results of the in vitro assay of the cytotoxic activity of the PG-1 fusion protein against HepG2 cells are shown in Fig. 4. The percentage of growth inhibition of the HepG2 cells by PG-1/6His at various concentrations was determined by the number of viable treated cells in comparison with the number of viable cells of the untreated controls. The results showed that PG-1/6His had a dose- and time-dependent inhibitory effect on cell growth.