Expression of porcine protegrin-1 in Pichia pastoris and its anticancer activity in vitro

Protegrin-1 (PG-1), a β-hairpin antimicrobial peptide (AMP), is amongst the shortest AMPs in sequence length while remaining active against a variety of microorganisms. The aim of this study was produce recombinant PG-1 and investigate its anticancer activity. A DNA sequence encoding the mature PG-1, fused with a 6His-tag, was cloned into the pPICZα-A vector and transformed into Pichia pastoris. Expression was induced following culture for ~96 h with 1% methanol at 28°C, and ~15.6 mg PG-1 was expressed in 100 ml culture medium. Following purification using a Ni-chelating Sepharose column, ~20 mg pure active PG-1 was obtained from 500 ml culture broth supernatant. The expressed PG-1/6His exhibited strong dose- and time-dependent anticancer activity against HepG2 cells in vitro.


Introduction
Antimicrobial peptides (AMPs) are significant components of the innate immune systems of numerous animal species, where they act as an effective, largely non-discriminatory first line of defense against invading pathogens (1). A variety of AMPs have been identified from a variety of sources, ranging from plants and insects to lower vertebrates and mammals, as described in previous studies (2,3).
Protegrin-1 (PG-1) is a β-hairpin AMP of 18 amino acids (RGGRLCYCRRRFCVCVGR) that was originally isolated from porcine leukocytes (4). PG-1 is rich in cationic residues, such as arginine (Arg). The amphipathic characteristic of this peptide enables it to interact with the membranes of pathogens and kill the pathogen by releasing its cellular contents (5). Studies have revealed a number of the key steps in the action of protegrin, including: Protegrin monomers dimerize in various types of lipid environment; protegrin peptides interact strongly with lipid bilayer membranes, particularly those that contain anionic lipids; and protegrins form pores in lipid bilayers, which results in uncontrolled ion transport and may be a key factor in bacterial death (1). PG-1 is considered as a potential pharmaceutical agent as it has shown a broad range of antimicrobial activities against gram-positive and gram-negative bacteria, including Escherichia coli (E. coli), Pseudomonas aeruginosa and Neisseria gonorrhoeae (5)(6)(7).
For pharmaceutical applications, a large quantity of AMP is required. Chemical synthesis of AMPs is not economically practical, particularly for the production of long peptides. Thus, a biological expression system would be an improved alternative method of production (2). Numerous biological expression systems have been introduced for the economical production of AMPs (8). For the production of recombinant AMPs, E. coli-based systems are well established (8,9) and Pichia pastoris (P. pastoris) expression systems are frequently used (10)(11)(12)(13)(14). However, each system has limitations, including poor recovery yields, proteolysis of products, low expression levels, toxicity of the product to host cells, and occasionally an absence of the post-translational modifications required for the biological activity of the AMPs (2,8,15).
The aim of the present study was to express the antimicrobial peptide PG-1 in P. pastoris X-33, isolate the recombinant product and investigate its anticancer activity. Design and synthesis of the PG-1 nucleotide sequence. 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).

Construction of the expression vectors.
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'-ATC AGG CGC CAT TCG CCA TTC-3') and pUC57-(5'-CAG GTT CCC GAC TGG AAAG-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 ddH 2 O. The PCR procedure involved 4 min predenaturation at 95℃; 30 sec denaturation at 94℃, 30 sec annealing at 53℃ and 30 sec extension at 72℃ for 30 cycles; followed by 3 min extension at 72℃. 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.
Heterologous expression of recombinant PG-1 in P. pastoris X-33. 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, 4x10 -5 % biotin and 1% glycerol) for 24 h at 28˚C. When the cell density reached ~0.6 at , the cells were harvested by centrifugation at 3,000 x 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, 4x10 -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 x 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).
Purification of peptide PG-1 and concentration determination. The expression supernatant was applied to a Ni-chelating Sepharose column (1.6x10 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 NaH 2 PO 4 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.
In vitro assay for cytotoxic activity. The cytotoxicity of the purified PG-1 fusion protein was determined by a tetrazolium (MTT) assay (18). The HepG2 cells (3x10 3 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.

Construction of the recombinant plasmid pPICZα-A-PG-1.
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.

Recombinant pPICZα-A-PG-1 expression and purification.
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.
Cytotoxic activity of PG-1 against HepG2 cells. 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.

Discussion
PG-1 (H 3 N + -RGGRLCYCRRRFCVCVGR-CONH 2 ), with a high content of positively charged Arg and cysteine (Cys) residues, has a β-hairpin structure that is stabilized by disulfide bonds linking Cys-6 and Cys-15, and Cys-8 and Cys-13 (19,20). Its rigid structure separates the hydrophobic and hydrophilic residues of the peptide, resulting in PG-1 having an amphipathic nature that is common to numerous other types of AMP (1,21). PG-1 is highly cationic (charge of 7+) at physiological pH, which is essential for its ability to bind strongly to bacterial cell membranes. It is the amphipathic characteristic of PG-1 that enables it to interact with the membranes of pathogens (4). Protegrin monomers dimerize in various types of lipid environment (1,4,19,21,22). However, the characteristics of the protegrin peptide biologically expressed in the present study, and whether fusion to 6His affects its structure and function are unknown. These factors require investigation in further studies.
The methylotrophic yeast P. pastoris has been utilized widely as a heterologous gene expression system. Although PG-1 has been successfully expressed in E. coli (8), the expression levels that have been achieved are lower than those obtained for P. pastoris-derived PG-1 in the present study. Furthermore, certain recombinant AMPs produced in P. pastoris have stronger activity than those produced in E. coli, including crab AMP scygonadin (12), plant defensin corn 1 (23), human secretory leukocyte protease inhibitor (24) and non-specific lipid-transfer proteins (25).
In order to facilitate the purification of PG-1 in the present study, a 6His-tag was introduced at the C-terminus of PG-1. A previous study has observed that Mdcec/6His has the same levels of activity against bacteria as those of Mdcec, and has slightly increased activity levels against fungi (11). P. pastoris-derived scygonadin/6His has been effectively expressed with higher activity levels against bacteria than those of scygonadin (12). In the present study, the expressed PG-1/6His demonstrated strong dose-and time-dependent anticancer activity against HepG2 cells.