Abstract
Interleukin-37 is a newly discovered cytokine that plays a pivotal role in suppressing innate inflammation and acquired immunity. We have recently expressed both the mature(mat-) and pro-forms of human IL-37b in plants and demonstrated that while both forms of the plant-made hIL-37b are functional, pmat-hIL37b exhibited significantly greater activity than ppro-IL-37b. Compared to ppro-hIL-37b, on the other hand, the expression level of pmat-hIL-37b was substantially lower (100.5 µg versus 1.05 µg/g fresh leaf mass or 1% versus 0.01% TSP). Since the difference between ppro-hIL-37b and pmat-hIL-37b is that ppro-hIL-37b contains a signal sequence not cleavable by plant cells, we reasoned that this signal sequence would play a key role in stabilizing the ppro-hIL-37b protein. Here, we describe a novel approach to enhancing pmat-hIL-37b production in plants based on incorporation of a gene sequence encoding tobacco etch virus (TEV) protease between the signal peptide and the mature hIL-37b, including a TEV cleavage site at the C-termini of TEV protease. The rationale is that when expressed as a sp-TEV-matIL-37b fusion protein, the stabilizing properties of the signal peptide of pro-hIL-37b will be awarded to its fusion partners, resulting in increased yield of target proteins. The fusion protein is then expected to cleave itself in vivo to yield a mature pmat-hIL-37b. Indeed, when a sp-TEV-matIL-37b fusion gene was expressed in stable-transformed plants, a prominent band corresponding to dimeric pmat-hIL-37b was detected, with expression yields reaching 42.5 µg/g fresh leaf mass in the best expression lines. Bioassays demonstrated that plant-made mature pmat-hIL-37b is functional.
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Acknowledgements
This research was supported by funding from the National Natural Science Foundation of China (Grant number 32072588, U22A20495) to Aoxue Wang.
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YZ and NA participated in project planning and experimental design and performed majority experiments. ZM, JZ, NL performed tobacco genetic transformation, immunoblotting, ELISA and protein affinity purification. YZ and MF performed vector construction and statistical analysis of experimental data. AW and SM wrote the manuscript. AW and SM designed and guided intellectually all processes of the work. All authors have read and approved the manuscript.
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Zhang, Y., Alqazlan, N., Meng, Z. et al. A novel approach to achieving more efficient production of the mature form of human IL-37 in plants. Transgenic Res 32, 279–291 (2023). https://doi.org/10.1007/s11248-023-00351-z
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DOI: https://doi.org/10.1007/s11248-023-00351-z