Expression And Biological Activity Evaluation Of Procine Protegrin-1

Antimicrobial peptides (AMPs) are important components of the innate immunesystems of countless animal species, where they act as an effective, largelynon-discriminatory first line of defense against invading pathogens. A great variety ofantimicrobial peptides have been discovered from various sources, ranging from plantsand insects to lower vertebrates and mammals as described in recent reviews.Protegrin-1(PG-1) is a β-hairpin antimicrobial peptide of18amino acids(RGGRL CYCRR RFCVC VGR) that was originally isolated from porcine leukocytes.PG-1are rich in cationic residues such as arginine. It is the amphipathic characteristicof the peptides that enables them to interact with the pathogen membrane and kill thepathogen by releasing its cellular contents. Experimental investigations have shed lighton many of the mechanism in the action of protegrin: protegrin monomers are knownto dimerize in various lipid environments; protegrin peptides interact strongly withlipid bilayer membranes, particularly anionic lipids; protegrins have been shown toform pores in lipid bilayers, which results in uncontrolled ion transport and may be akey factor in bacterial death. PG-1is considered as a potential pharmaceuticalagent,due to it has a broad range of antimicrobial activity against both Gram-positiveand Gram-negative bacteria.In this study, antimicrobial peptide PG-1was expressed in P. pastoris X-33. Therecombinant antimicrobial peptide was purified and its antimicrobial and anticanceractivity was confirmed.Based on the primary amino acid sequences of the mature peptide and accordingto codon preference of P. pastoris X-33, the gene sequence encoding PG-1. An Xho Irestriction enzyme site was introduced to allow inframe cloning into the α-factorsecretion signal of the pPICZa-PG-1shuttle vector, a sequence which encoding a Kex2cleavage site (LEKR) and a6His-tag coding sequence were added upstream anddownstream of the PG-1sequence, respectively. A termination codons of PG-1was added at the C-terminus, neighbour with a Xba I restriction enzyme site. The DNAsequence encoding the mature PG-1, fused with a6His-tag, was cloned into thepPICZa-A vector, Plasmid pPICZa-A and pPICZa-PG-1was digested by Sac Irespectively and transformed into P. pastoris X-33using electroporation methodand.PCR was performed to identify the positive transformants.The positive P. pastoris transformants were selected and inoculated into BMGYmedium for24h at30℃. When the cell density reached about0.6at OD600. The cellswere harvested by centrifugation and resuspended to an OD600of1.0in BMMYmedium. then, the cell were cultured for4~5days at28℃in a flask and addingmethanol to a final concentration of1%every24h. The fusion protein in thesupernatant was analyzed by Tricine-sodium-dodecyl-sulfate-polyacrylamide gelelectrophoresis (Tricine-SDS-PAGE). The protein concentration was determined usingBradrord method with BSA as a standard. The supernatant was loaded into a nickelchelating Sepharose column (1.6×10cm), The peak of PG-1was determined byfractions analysis of15%Tricine-SDS-PAGE, fractions containing PG-1was collected.The protein concentration of purified PG-1was determined using Bio-Rad dye agentwith BSA as a standard. Then the purified peptide was lyophilized and stored at20℃until needed.The minimal growth inhibition concentration (MIC) by a liquid growth inhibitionassay. Ten microliters from each dilution were distributed in a96-well polypropylenemicrotiter plate, and100μL of a suspension of mid-log bacteria (2~7×105CFU/mL)was added to each well. After16h incubation, The antibacterial effect was evaluatedby measuring OD600.The cytotoxicity of purified PG-1was determined by a tetrazolium(MTT) assay.Hep G2, EC109and MGC803Cells (1×103/well) were plated in100μL ofmedium/well in96-well plates (Costar Corning, USA). After overnight incubation,purified PG-1was added in various concentrations (10,20,40,80and160μg/mL).three wells were included in each concentration. After treatment with purified PG-1for1days,20μL of5mg/mL MTT (pH4.7) was added to each well and cultivated foranother4h. Then the supernatant fluid was removed,150μL DMSO was added intoeach sample and samples were shaken for10min. Absorbance at490nm was measuredwith a microplate reader (Bio-Rad, USA) using wells without cells as blanks. Threeindependent experiments were performed.The recombinant plasmid pPICZa-A-PG-1was constructed and was identified by restriction enzyme analysis and DNA sequencing. The identified recombinant plasmidswere transformed into P. pastoris X-33. The analysis of Tricine-SDS-PAGE showedthat the PG-1was successfully expressed and mainly in the supernatant, withmolecular weights corresponding with it’s theory values. After purification by a nickelchelating sepharose column, about10mg pure active PG-1was obtained from500mLculture broth supernatant. The purified PG-1showed a broader antibacterial spectrum,The MIC results indicated that E.coli and Bacillus subtilis were sensitive to thepurified PG-1at low concentration. The expressed PG-1has strong dose-dependentanticancer activity against HepG2, EC109and MGC803cells.This study would provide reference for the feasibility of using P. Pastorisexpression system to produce PG-1, and lay a good foundation for screening newantimicrobial drug.