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Enhancement of anti-tumor activity in melanoma using arginine deiminase fused with 30Kc19α protein

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Abstract

Arginine deiminase (ADI) is a microbial-derived enzyme which catalyzes the conversion of l-arginine into l-citrulline. ADI originating from Mycoplasma has been reported to present anti-tumor activity against arginine-auxotrophic tumors, including melanoma. Melanoma cells are sensitive to arginine depletion due to reduced expression of argininosuccinate synthase 1 (ASS1), a key enzyme for arginine biosynthesis. However, clinical applications of recombinant ADI for melanoma treatment present some limitations. Since recombinant ADI is not human-derived, it shows instability, proteolytic degradation, and antigenicity in human serum. In addition, there is a problem of drug resistance issue due to the intracellular expression of once-silenced ASS1. Moreover, recombinant ADI proteins are mainly expressed as inclusion body forms in Escherichia coli and require a time-consuming refolding process to turn them back into active form. Herein, we propose fusion of recombinant ADI from Mycoplasma hominis and 30Kc19α, a cell-penetrating protein which also increases stability and soluble expression of cargo proteins, to overcome these problems. We inserted matrix metalloproteinase-2 cleavable linker between ADI and 30Kc19α to increase enzyme activity in melanoma cells. Compared to ADI, ADI-LK-30Kc19α showed enhanced solubility, stability, and cell penetration. The fusion protein demonstrated selective cytotoxicity and reduced drug resistance in melanoma cells, thus would be a promising strategy for the improved efficacy in melanoma treatment.

Key points

• Fusion of ADI with 30Kc19α enhances soluble expression and productivity of recombinant ADI in E. coli

• 30Kc19α protects ADI from the proteolytic degradation by shielding effect, helping ADI to remain active

• Intracellular delivery of ADI by 30Kc19α overcomes ADI resistance in melanoma cells by degrading intracellularly expressed arginine

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Data availability

The data supporting the findings of this study are available within the article and its supplementary materials.

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Funding

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2020R1A4A3078645, 2021R1C1C1014606).

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Author notes

  1. Haein Lee and Geunhwa Park contributed equally to this work.

Authors and Affiliations

  1. School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, Seoul, Republic of Korea

    Haein Lee, Seulha Kim & Tai Hyun Park

  2. Interdisciplinary Program in Bioengineering, Seoul National University, Seoul, Republic of Korea

    Geunhwa Park & Tai Hyun Park

  3. Department of Bioengineering, Hanyang University, Seoul, Republic of Korea

    Boram Son & Hee Ho Park

  4. Department of Biomedical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan, Republic of Korea

    Jinmyoung Joo

  5. Education and Research Group for Biopharmaceutical Innovation Leader, Hanyang University, Seoul, Republic of Korea

    Hee Ho Park

  6. BioMax/N-Bio Institute, Institute of Bioengineering, Seoul National University, Seoul, Republic of Korea

    Tai Hyun Park

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Contributions

HL and GP collected data and evidence. HHP and THP conceived and designed the study. HL, GP, and HHP wrote the manuscript. HL drew the figures. HL, GP, SK, BS, JJ, and HHP directed and validated the data analysis. All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.

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Correspondence to Hee Ho Park or Tai Hyun Park.

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Lee, H., Park, G., Kim, S. et al. Enhancement of anti-tumor activity in melanoma using arginine deiminase fused with 30Kc19α protein. Appl Microbiol Biotechnol 106, 7531–7545 (2022). https://doi.org/10.1007/s00253-022-12218-0

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