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.

Interleukin-37 是一种新发现的细胞因子，在抑制先天性炎症和获得性免疫方面发挥着关键作用。我们最近在植物中表达了人 IL-37b 的成熟形式 (mat-) 和前形式，并证明虽然植物制造的 hIL-37b 的两种形式均具有功能，但 pmat-h​​IL37b 的活性明显高于 ppro-IL -37b。另一方面，与 ppro-hIL-37b 相比，pmat-h​​IL-37b 的表达水平显着降低（100.5 µg 与 1.05 µg/g 新鲜叶质量或 1% 与 0.01% TSP）。由于 ppro-hIL-37b 和 pmat-h​​IL-37b 之间的差异在于 ppro-hIL-37b 含有植物细胞不可切割的信号序列，因此我们推断该信号序列在稳定 ppro-hIL-37b 中发挥关键作用。 37b 蛋白质。在这里，我们描述了一种增强植物中 pmat-h​​IL-37b 产量的新方法，该方法基于在信号肽和成熟 hIL-37b 之间掺入编码烟草蚀纹病毒 (TEV) 蛋白酶的基因序列，包括位于TEV 蛋白酶的 C 末端。其基本原理是，当表达为 sp-TEV-matIL-37b 融合蛋白时，pro-hIL-37b 信号肽的稳定特性将赋予其融合伴侣，从而提高靶蛋白的产量。然后，预计融合蛋白会在体内裂解自身，产生成熟的 pmat-h​​IL-37b。事实上，当sp-TEV-matIL-37b融合基因在稳定转化的植物中表达时，检测到对应于二聚体pmat-h​​IL-37b的显着条带，在最佳表达中表达产量达到42.5μg/g新鲜叶质量线。生物测定证明植物制造的成熟 pmat-h​​IL-37b 具有功能性。