title: Design, expression, and characterization of a novel targeted plectasin against methicillin-resistant Staphylococcus aureus.
abstract: A novel specifically targeted antimicrobial peptide (STAMP) that was especially effective against methicillin-resistant Staphylococcus aureus (MRSA) was designed by fusing the AgrD1 pheromone to the N-terminal end of plectasin. This STAMP was named Agplectasin, and its gene was synthesized and expressed in Pichia pastoris X-33 via pPICZαA. The highest amount of total secreted protein reached 1,285.5 mg/l at 108 h during the 120-h induction. The recombinant Agplectasin (rAgP) was purified by cation exchange chromatography and hydrophobic exchange chromatography; its yield reached 150 mg/l with 94 % purity. The rAgP exhibited strong bactericidal activity against S. aureus but not Staphylococcus epidermidis or other types of tested bacteria. A bactericidal kinetics assay showed that the rAgP killed over 99.9 % of tested S. aureus (ATCC 25923 and ATCC 43300) in both Mueller-Hinton medium and human blood within 10 h when treated with 4× minimal inhibitory concentration. The rAgP caused only approximately 1 % hemolysis of human blood cells, even when the concentration reached 512 μg/ml, making it potentially feasible as a clinical injection agent. In addition, it maintained a high activity over a wide range of pH values (2.0-10.0) and demonstrated a high thermal stability at 100 °C for 1 h. These results suggested that this STAMP has the potential to eliminate MRSA strains without disrupting the normal flora.
authors.affiliation: Key Laboratory of Feed Biotechnology, Ministry of Agriculture, Beijing 100081, China.
authors.forename: Ruoyu
authors.initials: R
authors.lastname: Mao
authors.forename: Da
authors.initials: D
authors.lastname: Teng
authors.forename: Xiumin
authors.initials: X
authors.lastname: Wang
authors.forename: Di
authors.initials: D
authors.lastname: Xi
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhang
authors.forename: Xiaoyuan
authors.initials: X
authors.lastname: Hu
authors.forename: Yalin
authors.initials: Y
authors.lastname: Yang
authors.forename: Jianhua
authors.initials: J
authors.lastname: Wang
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism* / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D055624:Methicillin-Resistant Staphylococcus aureus / Q000187:drug effects*; D008826:Microbial Sensitivity Tests; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D010456:Peptides, Cyclic / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*
chemical_list: C414532:AgrD protein, Staphylococcus; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D010455:Peptides; D010456:Peptides, Cyclic; D011993:Recombinant Fusion Proteins; C504857:plectasin