Production and purification of tag-free recombinant human acid sphingomyelinase in Nicotiana benthamiana
Issued Date
2025-11-01
Resource Type
ISSN
07217714
eISSN
1432203X
Scopus ID
2-s2.0-105019711656
Journal Title
Plant Cell Reports
Volume
44
Issue
11
Rights Holder(s)
SCOPUS
Bibliographic Citation
Plant Cell Reports Vol.44 No.11 (2025)
Suggested Citation
Panyawechamontri K., Kajiura H., Misaki R., Fujiyama K. Production and purification of tag-free recombinant human acid sphingomyelinase in Nicotiana benthamiana. Plant Cell Reports Vol.44 No.11 (2025). doi:10.1007/s00299-025-03618-3 Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/112842
Title
Production and purification of tag-free recombinant human acid sphingomyelinase in Nicotiana benthamiana
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Abstract
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.