Abstract
PK34 is a D29 mycobacteriophage-derived anti-microbial peptide (AMP) with anti-Mycobacterium tuberculosis activity. It is expected to become an auxiliary drug for the treatment of M. tuberculosis infection, or as a template for the development of anti-M. tuberculosis drugs. The focus of this paper is to obtain recombinant PK34 by a novel method of prokaryotic expression and purification by affinity chromatography. The minimum inhibitory concentration (MIC) of recombinant PK34 was better than that of synthetic PK34 as measured by the microplate-based Alamar Blue assay (MABA). In order to further compare the different anti-bacterial effects of PK34 obtained by the two methods on M. tuberculosis, the bacterial changes after drug incubation were observed at the microscopic level by transmission electron microscopy (TEM). In order to apply PK34 to clinical treatment earlier in the future, this paper tested the maximum non-toxic concentration of recombinant PK34 to the two most studied immune cells, RAW264.7 and THP-1, through cytotoxicity experiments. The maximum non-toxic concentration was the same as the MIC of recombinant PK34 to M. tuberculosis H37Rv, and both were 12.5 μg/mL. The monoclonal antibodies against PK34 and their hybridoma cell lines were prepared using recombinant PK34 as the antigen. Next, we obtained the gene sequence of the monoclonal antibody, which was prepared for the basic research of PK34 in M. tuberculosis treatment. In addition, the possible molecular docking mode between PK34 and trehalose-6,6-dimycolate (TDM) was predicted by AI simulation. To sum up, this paper provides a new idea for the birth of more new AMPs of the same type as PK34 in the future.
Key points
• Design and prepare a novel recombinant PK34 anti-microbial peptide.
• Recombinant PK34 has higher purity and anti-bacterial activity than synthetic PK34.
• The monoclonal antibody against recombinant PK34 was prepared and sequenced.
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Data availability
The data sets used for the current study are available from the corresponding author upon reasonable request.
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Acknowledgements
The authors would like to thank the faculty and staff of the Specialized lab for medical examination of Weifang Medical University.
Funding
This work was supported by grants from the National Natural Science Foundation of China (Grant Nos. 81802054 and 81902170). Support program for Youth Innovation Technology in Colleges and Universities of Shandong Province of China (Grant No. 2021KJ106). The Tai-Shan Scholar Program from Shandong Province (Grant No. tsqn202103116).
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Conceived and designed the experiments: QL and JQW; performed the experiments: JQW, TXY, and XYH; analyzed the data: QL, JQW, HL, and TXY; prepared figures and/or tables: JQW and TXY; drafted manuscripts: QL, JQW, ZJY, and WG; and revised the manuscript: QL, JQW, ZJY, and WG. All the authors approved the submitted manuscript.
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All the experiments involving mice were performed in accordance with the Chinese National Laboratory Animal-Guideline for Ethical Review of Animal Welfare and approved by the Institutional Animal Care and Use Committee of Weifang Medical University. All the applicable international, national, and institutional guidelines for the care and use of animals were strictly followed. The study was approved by the Ethics Committee of Weifang Medical University, and all the research methods were in compliance with the relevant regulations and guidelines.
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Wang, J., Yuan, T., He, X. et al. Production, characterization, and application of phage-derived PK34 recombinant anti-microbial peptide. Appl Microbiol Biotechnol 107, 163–174 (2023). https://doi.org/10.1007/s00253-022-12306-1
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DOI: https://doi.org/10.1007/s00253-022-12306-1