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Synthesis of secretory leukocyte protease inhibitor using cell-free protein synthesis system

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Abstract

Secretory leukocyte protease inhibitor (SLPI) functions as a protease inhibitor that modulates excessive proteolysis in the body, exhibits broad-spectrum antimicrobial activity, regulates inflammatory responses, and plays an important role in the innate immunity. The purpose of the study was to artificially synthesize a SLPI, an antimicrobial peptide, and investigate its effect on antimicrobial activity against Porphyromonas gingivalis and interleukin-6 (IL-6) production. SLPI protein with a molecular weight of approximately 13 kDa was artificially synthesized using a cell-free protein synthesis (CFPS) system and investigated by western blotting and enzyme-linked immunosorbent assay (ELISA). Disulfide bond isomerase in the protein synthesis mixture increased the amount of SLPI synthesized. The synthesized SLPI (sSLPI) protein was purified and its antimicrobial activity was investigated based on the growth of Porphyromonas gingivalis and bacterial adhesion to oral epithelial cells. The effect of sSLPI on IL-6 production in human periodontal ligament fibroblasts (HPLFs) was examined by ELISA. Our results showed that sSLPI significantly inhibited the growth of Porphyromonas gingivalis and bacterial adhesion to oral epithelial cells and further inhibited IL-6 production by HPLFs. These results suggested that SLPI artificially synthesized using the CFPS system may play a role in the prevention of periodontal diseases through its antimicrobial and anti-inflammatory effects.

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All data supporting the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank GeneFrontier Co., Ltd. (Chiba, Japan) for supporting the design of template DNA sequences with AT-rich codons for SLPI synthesis.

Funding

This study was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (grant numbers: 17H04418, 20K09941, 20K23083, and 22K17040).

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Authors and Affiliations

  1. Department of Oral Microbiology, Tokushima University Graduate School of Biomedical Sciences, 3-18-15, Kuramoto, Tokushima, 770-8504, Japan

    Yuka Hiroshima & Akikazu Murakami

  2. Department of Periodontology and Endodontology, Tokushima University Graduate School of Biomedical Sciences, Tokushima, Japan

    Rie Kido, Jun-ichi Kido & Mika Bando

  3. Department of Oral Healthcare Promotion, Tokushima University Graduate School of Biomedical Sciences, Tokushima, Japan

    Kaya Yoshida

  4. Institute of Advanced Medical Sciences, Tokushima University, Tokushima, Japan

    Yasuo Shinohara

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  1. Yuka Hiroshima
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  5. Kaya Yoshida
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Contributions

Yuka Hiroshima: funding acquisition, investigation, methodology, validation, writing –original draft, writing—review and editing. Rie Kido: investigation, writing—original draft, writing—review and editing. Jun-ichi Kido: conceptualization, funding acquisition, investigation, methodology, validation; writing- original draft, writing—review and editing. Mika Bando: investigation, writing—original draft, writing—review and editing. Kaya Yoshida: investigation, writing—original draft, writing—review and editing. Akikazu Murakami: investigation, writing—original draft, writing—review and editing. Yasuo Shinohara: writing—original draft, writing—review and editing.

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Correspondence to Yuka Hiroshima.

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Hiroshima, Y., Kido, R., Kido, Ji. et al. Synthesis of secretory leukocyte protease inhibitor using cell-free protein synthesis system. Odontology (2024). https://doi.org/10.1007/s10266-024-00910-8

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