title: High expression of a plectasin-derived peptide NZ2114 in Pichia pastoris and its pharmacodynamics, postantibiotic and synergy against Staphylococcus aureus.
abstract: NZ2114, a new variant of plectasin, was overexpressed in Pichia pastoris X-33 via pPICZαA for the first time. The total secreted protein of fermentation supernatant reached 2,390 mg/l (29 °C) and 2,310 mg/l (25 °C), and the recombinant NZ2114 (rNZ2114) reached 860 mg/l (29 °C) and 1,309 mg/l (25 °C) at 96 h induction in a 5-l fermentor, respectively.The rNZ2114 was purified by cation exchange chromatography, and its yield was 583 mg/l with 94.8 % purity. The minimal inhibitory concentration (MIC) of rNZ2114 to four ATCC strains of Staphyloccocus aureus was evaluated from 0.028 to 0.90 μM. Meanwhile, it showed potent activity (0.11-0.90 μM) to 20 clinical isolates of MRSA. The rNZ2114 killed over 99.9 % of tested S. aureus (ATCC 25923 and ATCC 43300) in Mueller-Hinton medium within 6 h when treated with 4 × MIC. The postantibiotic effect of rNZ2114 to S. aureus ATCC 25923 and ATCC 43300 was 18.6-45.6 and 1.7-3.5 h under 1×, 2×, and 4× MIC, respectively. The fractional inhibitory concentration index (FICI) indicated a synergistic effect between rNZ2114 and kanamycin, streptomycin, and vancomycin against S. aureus ATCC 25923 (FICI = 0.125), and additivity between rNZ2114 and ampicillin, spectinomycin (FICI = 0.625), respectively. To S. aureus ATCC 43300 [methicillin-resistant S. aureus (MRSA)], rNZ2114 showed a synergistic effect (FICI = 0.125-0.3125) with kanamycin, ampicillin, streptomycin, and vancomycin, and antagonism with spectinomycin (FICI = 8.0625). The rNZ2114 caused only less than 0.1 % hemolytic activity in the concentration of 128 μg/ml, and showed a good thermostability from 20 to 80 °C. In addition, it exhibited the highest activity at pH 8.0. These results suggested that large-scale production of NZ2114 is feasible using the P. pastoris expression system, and it could be a new potential antimicrobial agent for the prevention and treatment of S. aureus especially for MRSA infections.
authors.affiliation: Key Laboratory of Feed Biotechnology, Ministry of Agriculture, Beijing, 100081, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhang
authors.forename: Da
authors.initials: D
authors.lastname: Teng
authors.forename: Ruoyu
authors.initials: R
authors.lastname: Mao
authors.forename: Xiumin
authors.initials: X
authors.lastname: Wang
authors.forename: Di
authors.initials: D
authors.lastname: Xi
authors.forename: Xiaoyuan
authors.initials: X
authors.lastname: Hu
authors.forename: Jianhua
authors.initials: J
authors.lastname: Wang
mesh_terms: D000900:Anti-Bacterial Agents / Q000302:isolation & purification / Q000494:pharmacology*; D002852:Chromatography, Ion Exchange; D004357:Drug Synergism; D015870:Gene Expression; D008826:Microbial Sensitivity Tests; D050296:Microbial Viability / Q000187:drug effects; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D013211:Staphylococcus aureus / Q000187:drug effects*
chemical_list: D000900:Anti-Bacterial Agents; D010455:Peptides; D011994:Recombinant Proteins; C504857:plectasin