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Design, expression, and characterization of a novel targeted plectasin against methicillin-resistant Staphylococcus aureus

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An Erratum to this article was published on 12 March 2013

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.

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Acknowledgments

The authors wish to acknowledge Prof. Shi Xianming, Ph.D., in the Bor Luh Food Safety Center, Shanghai Jiaotong University, for donating all the MRSA clinical isolates, and Prof. Yang Fuquan, Ph.D., in the Proteomics Platform Laboratory at the Institute of Biophysics, Chinese Academy of Sciences, for his coordination of the MALDI-TOF MS analysis. This study is supported by the National Natural Science Foundation of China (nos. 30771574, 30810303084, 30972125, and 31001026) and the Beijing Natural Science Foundation (nos. 5062031 and 5093030).

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Authors and Affiliations

  1. Key Laboratory of Feed Biotechnology, Ministry of Agriculture, Beijing, 100081, China

    Ruoyu Mao, Da Teng, Xiumin Wang, Di Xi, Yong Zhang, Xiaoyuan Hu, Yalin Yang & Jianhua Wang

  2. Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences, 12 Zhongguancun Nandajie St., Haidian District, Beijing, 100081, People’s Republic of China

    Ruoyu Mao, Da Teng, Xiumin Wang, Di Xi, Yong Zhang, Xiaoyuan Hu & Jianhua Wang

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  1. Ruoyu Mao
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Correspondence to Jianhua Wang.

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Mao, R., Teng, D., Wang, X. et al. Design, expression, and characterization of a novel targeted plectasin against methicillin-resistant Staphylococcus aureus . Appl Microbiol Biotechnol 97, 3991–4002 (2013). https://doi.org/10.1007/s00253-012-4508-z

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