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Published on: August 11, 2018
Production, characterization, and application of phage-derived PK34 recombinant anti-microbial peptide
Jiaqi Wang1,2, Tingxun Yuan1,2, Xinyu He1,2
1Engineering Research Institute of Precision Medicine Innovation and Transformation of Infections Diseases, Weifang Medical University, Weifang, Shandong, 261053, People's Republic of China.
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.
Insights
This study developed recombinant PK34, an antimicrobial peptide effective against Mycobacterium tuberculosis. Recombinant PK34 demonstrated superior purity and antibacterial activity compared to synthetic versions, offering a promising new therapeutic avenue.
Area of Science:
- Microbiology
- Biotechnology
- Drug Discovery
Background:
- Mycobacterium tuberculosis infection remains a significant global health challenge.
- Antimicrobial peptides (AMPs) are emerging as potential therapeutic agents.
- PK34, a mycobacteriophage-derived AMP, shows promise for treating tuberculosis.
Purpose of the Study:
- To develop a novel method for producing recombinant PK34 using prokaryotic expression and affinity chromatography.
- To compare the antimicrobial activity and characteristics of recombinant PK34 against synthetic PK34.
- To assess the safety and potential clinical applicability of recombinant PK34.
Main Methods:
- Prokaryotic expression and affinity chromatography for recombinant PK34 purification.
- Microplate-based Alamar Blue assay (MABA) for minimum inhibitory concentration (MIC) determination.
- Transmission electron microscopy (TEM) for observing bacterial morphological changes.
- Cytotoxicity assays on RAW264.7 and THP-1 immune cells.
- Monoclonal antibody production and sequencing.
- AI-driven molecular docking simulations.
Main Results:
- Recombinant PK34 was successfully expressed and purified with high purity.
- Recombinant PK34 exhibited enhanced antimicrobial activity against Mycobacterium tuberculosis compared to synthetic PK34.
- The maximum non-toxic concentration of recombinant PK34 for immune cells was 12.5 μg/mL, matching its MIC.
- Monoclonal antibodies against PK34 were generated, providing tools for further research.
- AI simulation predicted a molecular docking mode between PK34 and trehalose-6,6-dimycolate (TDM).
Conclusions:
- Recombinant PK34 offers a superior alternative to synthetic PK34 for potential tuberculosis treatment.
- The established production and characterization methods pave the way for developing similar novel AMPs.
- Further research utilizing monoclonal antibodies and molecular insights can advance PK34's clinical application.
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