Panyawechamontri K.Kajiura H.Misaki R.Fujiyama K.Mahidol University2025-10-312025-10-312025-11-01Plant Cell Reports Vol.44 No.11 (2025)07217714https://repository.li.mahidol.ac.th/handle/123456789/112842Key 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.Agricultural and Biological SciencesArticleSCOPUS10.1007/s00299-025-03618-32-s2.0-1050197116561432203X