Xiong Yongao, Hirano Hiroto, Lane Nancy E, Nandi Somen, McDonald Karen A
Department of Chemical Engineering, University of California, Davis, CA, United States.
Center for the Utilization of Biological Engineering in Space (CUBES), Berkeley, CA, United States.
Front Bioeng Biotechnol. 2022 Sep 12;10:962292. doi: 10.3389/fbioe.2022.962292. eCollection 2022.
Microgravity-induced bone loss is a main obstacle for long term space missions as it is difficult to maintain bone mass when loading stimuli is reduced. With a typical bone mineral density loss of 1.5% per month of microgravity exposure, the chances for osteoporosis and fractures may endanger astronauts' health. Parathyroid Hormone or PTH (1-34) is an FDA approved treatment for osteoporosis, and may reverse microgravity-induced bone loss. However, PTH proteins requires refrigeration, daily subcutaneous injection, and have a short shelf-life, limiting its use in a resource-limited environment, like space. In this study, PTH was produced in an Fc-fusion form via transient expression in plants, to improve the circulatory half-life which reduces dosing frequency and to simplify purification if needed. Plant-based expression is well-suited for space medicine application given its low resource consumption and short expression timeline. The PTH-Fc accumulation profile in plant was established with a peak expression on day 5 post infiltration of 373 ± 59 mg/kg leaf fresh weight. Once the PTH-Fc was purified, the amino acid sequence and the binding affinity to its target, PTH 1 receptor (PTH1R), was determined utilizing biolayer interferometry (BLI). The binding affinity between PTH-Fc and PTH1R was 2.30 × 10 M, similar to the affinity between PTH (1-34) and PTH1R (2.31 × 10 M). Its function was also confirmed in a cell-based receptor stimulation assay, where PTH-Fc was able to stimulate the PTH1R producing cyclic adenosine monophosphate (cAMP) with an EC of (8.54 ± 0.12) x 10 M, comparable to the EC from the PTH (1-34) of 1.49 × 10 M. These results suggest that plant recombinant PTH-Fc exhibits a similar binding affinity and potency in a PTH1R activation assay compared to PTH. Furthermore, it can be produced rapidly at high levels with minimal resources and reagents, making it ideal for production in low resource environments such as space.
微重力引起的骨质流失是长期太空任务的主要障碍，因为当负荷刺激减少时，很难维持骨量。在微重力环境下，每月典型的骨矿物质密度损失为1.5%，骨质疏松和骨折的风险可能危及宇航员的健康。甲状旁腺激素或PTH(1 - 34)是一种经美国食品药品监督管理局(FDA)批准用于治疗骨质疏松症的药物，可能会逆转微重力引起的骨质流失。然而，PTH蛋白需要冷藏、每日皮下注射，且保质期短，这限制了它在资源有限的环境(如太空)中的使用。在本研究中，通过在植物中瞬时表达产生Fc融合形式的PTH，以延长循环半衰期，从而减少给药频率，并在需要时简化纯化过程。鉴于其低资源消耗和短表达时间线，基于植物的表达非常适合太空医学应用。在植物中建立了PTH - Fc积累曲线，在浸润后第5天达到峰值表达，为373±59mg/kg叶鲜重。一旦PTH - Fc被纯化，利用生物层干涉术(BLI)确定其氨基酸序列及其与靶标甲状旁腺激素1受体(PTH1R)的结合亲和力。PTH - Fc与PTH1R之间的结合亲和力为2.30×10⁻⁸M，与PTH(1 - 34)和PTH1R之间的亲和力(2.31×10⁻⁸M)相似。其功能也在基于细胞的受体刺激试验中得到证实，其中PTH - Fc能够刺激PTH1R产生环磷酸腺苷(cAMP)，其半数有效浓度(EC₅₀)为(8.54±0.12)×10⁻⁸M，与PTH(1 - 34)的EC₅₀ 1.49×10⁻⁸M相当。这些结果表明，与PTH相比，植物重组PTH - Fc在PTH1R激活试验中表现出相似的结合亲和力和效力。此外，它可以用最少的资源和试剂快速大量生产，使其成为在太空等低资源环境中生产的理想选择。
