Cost-effective downstream processing of recombinantly produced pexiganan peptide and its antimicrobial activity

Abstract

Antimicrobial peptides (AMPs) have significant potential as alternatives to classical antibiotics. However, AMPs are currently prepared using processes which are often laborious, expensive and of low-yield, thus hindering their research and application. Large-scale methods for production of AMPs using a cost-effective approach is urgently required. In this study, we report a scalable, chromatography-free downstream processing method for producing an antimicrobial peptide, pexiganan, using recombinant Escherichia coli (E. coli). The four helix bundle structure of the unique carrier protein DAMP4 was used to facilitate a simple and cheap purification process based on a selective thermochemical precipitation. Highly pure fusion protein DAMP4_var-pexiganan was obtained at high yield (around 24 mg per 800 mL cell culture with a final cultivation OD₆₀₀ ~ 2). The purification yield of DAMP4_var-pexiganan protein is increased twofold with a 72.9% of the protein recovery in this study as compared to the previous purification processes (Dwyer in Chem Eng Sci 105:12-21, 2014). The antimicrobial peptide pexiganan was released and activated from the fusion protein by a simple acid-cleavage. Isoelectric precipitation was then applied to separate the pexiganan peptide from the DAMP4_var protein carrier. The final yield of pure bio-produced pexiganan was 1.6 mg from 800 mL of bacterial cell culture (final cultivation OD₆₀₀ ~ 2). The minimum bactericidal concentration (MBC) test demonstrated that the bio-produced pexiganan has the same antimicrobial activity as chemically synthesized counterpart. This novel downstream process provides a new strategy for simple and probable economic production of antimicrobial peptides.

Keywords: Antimicrobial peptides; Carrier proteins; Minimum bactericidal concentration; Protein purification; Recombinant E. coli; Selective precipitation.

Fig. 1

Effect of NaCl concentrations (0–2 M) on the DNA precipitation using poly(ethyleneimine) (PEI) 0.5% (w/v) in Tris–HCl buffer (25 mM, pH 8). a SDS–PAGE analysis. Concentrations of (b) DAMP4_var-pexiganan protein and c DNA remaining in the solution after PEI addition

Fig. 2

Effect of poly(ethyleneimine) (PEI) concentrations (0.05–0.5% (w/v)) on the DNA precipitation in Tris–HCl buffer (25 mM, pH 8) containing 1 M NaCl

Fig. 3

Effect of Na₂SO₄ concentrations (0–1 M) on the precipitation of protein contaminants at 90 °C as shown by SDS–PAGE gel

Fig. 4

Effect of Na₂SO₄ concentrations (1.6–2.4 M) on the solubility of DAMP4_var-pexiganan protein. a SDS–PAGE analysis (S, supernatant; P, precipitate). b The concentrations of DAMP4_var-pexiganan protein in both the supernatant and the solubilized precipitate

Fig. 5

Characterization of DAMP4_var-pexiganan protein after the purification process as determined by using RP-HPLC

Fig. 6

Process flow diagram of the cleavage of pexiganan peptide from the protein carrier DAMP4_var

Fig. 7

a Effect of incubation time on the cleavage of DAMP4_var-pexiganan protein to yield the bio-produced pexiganan peptide. b RP-HPLC characterization of the solution composition after the cleavage and subsequent isoelectric precipitation of DAMP4_var protein

Fig. 8

Antimicrobial activity of the bio-produced pexiganan peptide against E. coli ATCC^® 25922™ in comparison with controls as determined by the minimum bactericidal concentration (MBC) method. a The percentages of viable cells after MBC tests. b Photographs of E. coli growth on agar plates containing of: (a) water; (b) DAMP4 protein; (c) DAMP4_var-pexiganan protein; (d) synthetic pexiganan peptide; and (e) bio-produced pexiganan peptide. The concentration of all protein/peptide samples was 16 µg/mL

Fig. 9

Process flow diagram of the purification of DAMP4_var-pexiganan protein