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Key message <p>Tag-free, functional human acid sphingomyelinase was successfully produced in a plant-based system. Apoplastic wash fluid extraction improved downstream processing, and a two-step ion exchange chromatography enabled purification of plant-derived ASM.</p> Abstract <p>Acid sphingomyelinase (ASM) converts sphingomyelin into phosphocholine and ceramide, a process essential for various cellular functions. Given the relevance of ASM to human health and its potential as a therapeutic enzyme, the development of efficient recombinant production systems is of significant interest in biotechnology. We here developed a plant-based expression system for producing human ASM and targeted major limitations related to its purification. The purification was improved in two ways: by engineering a truncated ASM with a plant-derived secretion signal peptide and by utilizing apoplastic wash fluid extraction to improve the purification process. Recombinant ASM was produced in <i>N. benthamiana</i> as a functional protein using an <i>Agrobacterium</i>-mediated transient expression system. The recombinant ASM was then purified using a two-step ion exchange chromatography method, ensuring high purity. After purification, the ASM yield reached approximately 3.5 mg per kg of fresh leaf weight, with a yield of 49.14% and a 21.2-fold purification enhancement. The purified enzyme exhibited a specific activity of 128.18 ± 4.18 mU/mg, confirming that the plant-derived ASM was functionally active. This work represents the first successful production of human ASM in plants, along with the development of an optimized purification method. This achievement marks a significant step forward in overcoming the challenges associated with producing and purifying recombinant proteins in plant-based expression systems, paving the way for future therapeutic applications.</p>

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Production and purification of tag-free recombinant human acid sphingomyelinase in Nicotiana benthamiana

  • 期刊论文

  • 发表日期：

    2025年10月25日

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  • Kulachatr Panyawechamontri,
  • Hiroyuki Kajiura,
  • Ryo Misaki,
  • Kazuhito Fujiyama

摘要

Key message

Tag-free, functional human acid sphingomyelinase was successfully produced in a plant-based system. Apoplastic wash fluid extraction improved downstream processing, and a two-step ion exchange chromatography enabled purification of plant-derived ASM.

Abstract

Acid sphingomyelinase (ASM) converts sphingomyelin into phosphocholine and ceramide, a process essential for various cellular functions. Given the relevance of ASM to human health and its potential as a therapeutic enzyme, the development of efficient recombinant production systems is of significant interest in biotechnology. We here developed a plant-based expression system for producing human ASM and targeted major limitations related to its purification. The purification was improved in two ways: by engineering a truncated ASM with a plant-derived secretion signal peptide and by utilizing apoplastic wash fluid extraction to improve the purification process. Recombinant ASM was produced in N. benthamiana as a functional protein using an Agrobacterium-mediated transient expression system. The recombinant ASM was then purified using a two-step ion exchange chromatography method, ensuring high purity. After purification, the ASM yield reached approximately 3.5 mg per kg of fresh leaf weight, with a yield of 49.14% and a 21.2-fold purification enhancement. The purified enzyme exhibited a specific activity of 128.18 ± 4.18 mU/mg, confirming that the plant-derived ASM was functionally active. This work represents the first successful production of human ASM in plants, along with the development of an optimized purification method. This achievement marks a significant step forward in overcoming the challenges associated with producing and purifying recombinant proteins in plant-based expression systems, paving the way for future therapeutic applications.