Materials
Synthetic pexiganan peptide (GIGKFLKKAKKFGKAFVKILKK, MW 2477.19 Da) was custom synthesized by Genscript Corporation (Piscataway, NJ) with purity > 99%. Poly(ethyleneimine) (PEI) 50% (w/v) in water was purchased from Sigma-Aldrich (#P3143, Castle Hill, Australia). A stock solution of PEI 5% (w/v) at pH 8 was prepared by adding hydrochloric acid (HCl). Water with > 18.2 MΩ cm resistivity was obtained from a Milli-Q system with a 0.22 µm filter (Millipore, North Ryde, Australia). All chemicals were of analytical grade obtained from either Sigma-Aldrich or Merck (Frenchs Forest, Australia) and were used as received unless otherwise stated.
Expression of DAMP4var-pexiganan protein
Recombinant plasmid pET-48b(+) comprising a nucleotide sequence encoding DAMP4var-pexiganan protein (GenBank Accession Number: MG029580) (Protein Expression Facility, The University of Queensland) was transformed into chemically competent E. coli strain BL21(DE3) (Novagen Merck Bioscience, Darmstadt, Germany). The cells were streaked on a Luria–Bertani (LB) agar plate (15 g/L agar, 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl) and then incubated at 37°C overnight. A single colony selected from the plate was inoculated into 5 mL LB media (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl), followed by incubation at 37 °C, 180 rpm (Ratek, Boronia, Australia) overnight. Overnight culture (800 µL, OD600 ~ 2.5) was added into 800 mL of 2 × yeast extract and tryptone (2YT) media (16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl) and then incubated at 37 °C, 180 rpm until OD600 ~ 0.5. Protein expression was induced by adding isopropyl-b-d-thiogalactopyranoside (Astral Scientific, Taren Point, Australia) to a final concentration of 1 mM, and cells were further incubated at 37 °C, 180 rpm for 4 h (final OD600 ~ 2). Cells were harvested by centrifugation (4000×g, 4 °C, 20 min) and cell pellets were stored at − 20 °C for subsequent use. All bacterial-growth media were supplemented with 50 mg/L of kanamycin sulfate.
Optimization of DAMP4var-pexiganan purification
Cell pellet was resuspended in 40 mL of lysis buffer (25 mM Tris–HCl, pH 8) containing NaCl, and cells were lysed by sonication (Branson Ultrasonics, Danbury, CT) at an energy output of 60 W for 4 burst of 30 s and interspersed in an ice bath for 60 s.
DNA contaminants were removed from crude cell lysates by optimizing concentrations of NaCl and PEI added. To optimize NaCl concentration, NaCl (solid) was added into aliquots of cell lysate to final concentrations varied from 0.2 to 2 M, followed by addition of PEI to a final PEI concentration of 0.5% (w/v) with stirring at 4 °C for 60 min. To optimize PEI concentration, aliquots of PEI solution were added into aliquots of cell lysate containing 1 M NaCl to give final PEI concentrations ranging from 0.05 to 0.5% (w/v) with stirring at 4 °C for 60 min.
After removal of DNA from crude cell lysate, protein solution was collected by centrifugation (38,000×g, 4 °C, 20 min). Na2SO4 (solid) was added into aliquots of the supernatant to final concentrations ranging from 0.2 to 1 M with stirring at 90 °C for 30 min to optimize the precipitation of protein contaminants.
Subsequently, suspension was centrifuged (38,000×g, 25 °C, 20 min), and the resulting supernatant was added with Na2SO4 (solid) to give final Na2SO4 concentrations ranging from 1.6 to 2.4 M, followed by stirring at 35 °C for 60 min to allow optimization of the selective precipitation of DAMP4var-pexiganan protein.
The precipitate of DAMP4var-pexiganan protein was recovered by centrifugation (38,000×g, 35 °C, 20 min), and then washed with rinsing buffer (25 mM Tris–HCl, 1 M NaCl, pH 8) containing Na2SO4 at a concentration that retained the protein as a precipitate. Following centrifugation (38,000×g, 25 °C, 20 min), the precipitate was resuspended in solubilizing buffer (25 mM Tris–HCl, 1 M NaCl, pH 8), and then dialyzed against 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (25 mM, pH 7.5) using a dialysis tubing with SnakeSkin® pleated dialysis membrane (Thermo Fisher Scientific, North Ryde, Australia) with a molecular weight cut-off of 3.5 kDa. The protein solution after buffer exchange was subjected to the peptide-cleavage process.
Cleavage of pexiganan peptide from the DAMP4var protein carrier
HCl was added to the purified DAMP4var-pexiganan protein to a final pH of 4, and then incubated at 60 °C with incubation times varied from 1 to 48 h to optimize the cleavage of pexiganan peptide from DAMP4var-pexiganan protein. Subsequently, an aqueous solution of 2 M NaOH was added into the solution (final pH 6.8) and incubated at room temperature for 30 min to induce the precipitation of cleaved DAMP4var protein (theoretical pI 6.8). Supernatant containing pexiganan peptide was collected by centrifugation (38,000×g, 4 °C, 10 min), and then desalted against water by using an ÄKTA Explorer 10 system equipped with a 5-mL column of Sephadex G-25 resin (GE Healthcare, UK).
Analytical characterization
To determine DNA concentration, the protein samples (100 µL) were mixed with 1× SYBR® Safe (100 µL) (Life Technologies, Mulgrave, Australia) in a 96-well plate (Greiner Bio-One, Frickenhausen, Germany) for 5 min in a dark room, and then fluorescence intensities were acquired using Infinite® M200 Pro microplate reader (Tecan, Männedorf, Switzerland) at excitation and emission wavelengths of 502 and 530 nm, respectively. A standard curve of DNA (0–50 ng/mL) was constructed using the recombinant plasmid which was extracted and purified from E. coli XL1-Blue by using PureLink™ Quick Plasmid Miniprep (Thermo Fisher Scientific, North Ryde, Australia).
Protein samples were qualitatively analyzed by sodium dodecyl sulfate poly(acrylamide) gel electrophoresis (SDS-PAGE) using NuPAGE 4–12% Bis–Tris Precast Gels (Life Technologies, Mulgrave, Australia) mounted in a Bio-Rad XCell 3 system (Bio-Rad, Hercules, CA) with an aqueous buffer solution of 2-(N-morpholino)ethanesulfonic acid. Novex™ Sharp Pre-stained Protein Standard (Invitrogen, Carlsbad, CA) was used for the protein ladder.
Concentrations of proteins and peptides were quantitatively determined using reversed-phase high-performance liquid chromatography (RP-HPLC) equipped with a Jupiter C18 column (5 µm, 300 Å, 150 mm × 4.6 mm) (Phenomenex, Torrance, CA) and connected to an LC-10AVP series HPLC system (Shimadzu, Kyoto, Japan). Buffer A was 0.1% (v/v) trifluoroacetic acid (TFA) in water, and buffer B was 90% (v/v) acetonitrile and 0.1% (v/v) TFA in water. A linear gradient from 30 to 65% of buffer B was applied at a flow rate of 1 mL/min in 35 min and a detection wavelength was set at 214 nm.
Antimicrobial activity assay
The antimicrobial activity of the bio-produced pexiganan peptide was determined as compared to that of the controls (water, DAMP4 protein, DAMP4var-pexiganan protein, and synthetic pexiganan peptide) by using the minimum bactericidal concentration (MBC) method (Hu et al. 2010). Briefly, a single colony of E. coli ATCC® 25922™ (Manassas, VA) selected from a freshly streaked plate was inoculated into 5 mL Mueller-Hinton (MH) Broth (Becton–Dickinson, Sparks, MD) at 37 °C, 180 rpm, and then harvested at the exponential growth phase (OD600 ~ 0.5). After rinsing the cells twice by centrifugation (4000×g, 4 °C, 20 min), a standard cell suspension was prepared by resuspending the cell pellet in 0.9% NaCl solution to a final concentration of 107 colony-forming units (CFU) per mL (OD600 ~ 0.08). Protein/peptide samples (at final concentrations ranging from 1 to 32 µg/mL) as well as water were added into the standard cell suspensions to a final volume of 2 mL. Following incubation at 37 °C, 180 rpm for 2 h, the mixtures (diluted 10,000× in sterilized water) were each spread onto MH agar plates (MH Broth, 1.5% agar) and then incubated at 37 °C for overnight. The percentages of viable cells grown on the agar plates containing the protein/peptide samples were determined by counting the number of the colonies in comparison with the controls.