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Recombinant antimicrobial peptide hPAB-β expressed in Pichia pastoris, a potential agent active against methicillin-resistant Staphylococcus aureus

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Abstract

As a potential therapeutic agent, antimicrobial peptide has received increased attention in recent years. However, high-level expression of a small peptide with antimicrobial activity is still a challenging task. In this study, the coding sequence of antimicrobial peptide hPAB-β, a variant derived from human beta-defensin 2, was cloned into pPIC9K vector and transformed into Pichia pastoris. P. pastoris transformants harbored with multi-copy plasmids were screened by G418 selection. When the transformed cells were induced by methanol, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot, and matrix-assisted laser desorption ionization-time of flight mass spectrometry revealed recombinant hPAB-β products consisting of three protein species of 4,680.4, 4,485.3, and 4,881.9 Da at proportions of 58%, 36%, and 6%, respectively, which may be due to the incomplete processing of the fusion signal peptide of α-factor by the STE13 protease. Expressed hPAB-β was secreted into the culture medium at a level of 241.2 ± 29.5 mg/L. Purified hPAB-β with 95% homogeneity was obtained by 10 kDa membrane filtration followed by cation ion-exchange chromatography with a SP-Sepharose™ XL column. The two major protein species separated through a SOURCE™ 30RPC reverse phase chromatography column showed definite antimicrobial activities against Staphylococcus aureus. All 22 methicillin-resistant S. aureus (MRSA) isolates with multidrug resistance phenotype were sensitive to the recombinant hPAB-β with minimal inhibitory concentrations of 8–64 μg/ml. Our results show that the methylotrophic yeast-inducible system is suitable for high-level expression of active hPAB-β, and that expressed hPAB-β in P. pastoris may be a potential antimicrobial agent against MRSA infection.

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Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (30772061), Natural Science Foundation of Chongqing City (CSTC, 2005AB5201), and The “Eleven-Five” program of PLA (06G075). We thank Dr. Ling He at the Johns Hopkins University School of Medicine (USA) for the critical reading and modification of the manuscript.

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Authors and Affiliations

  1. Department of Microbiology, College of Medicine, Key Lab of Microbial Engineering Under the Educational Committee in Chongqing, Third Military Medical University, Chongqing, 400038, People’s Republic of China

    Zhijin Chen, Dongmei Wang, Yanguang Cong, Jing Wang, Junmin Zhu, Jie Yang, Zhen Hu, Xiaomei Hu, Yinling Tan, Fuquan Hu & Xiancai Rao

  2. Department of Microbiology, 30# Gaotanyan Street, Shapingba District, Chongqing, 400038, People’s Republic of China

    Xiancai Rao

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  1. Zhijin Chen
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  2. Dongmei Wang
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  3. Yanguang Cong
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  4. Jing Wang
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Correspondence to Xiancai Rao.

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Chen, Z., Wang, D., Cong, Y. et al. Recombinant antimicrobial peptide hPAB-β expressed in Pichia pastoris, a potential agent active against methicillin-resistant Staphylococcus aureus . Appl Microbiol Biotechnol 89, 281–291 (2011). https://doi.org/10.1007/s00253-010-2864-0

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