Department of Bioengineering and Technology, Faculty of Technology, Gauhati University, Guwahati, Assam 781014, India.
Department of Agricultural Biotechnology, Assam Agriculture University, Jorhat, Assam 785013, India.
J Biotechnol. 2020 Nov 10;323:302-312. doi: 10.1016/j.jbiotec.2020.07.012. Epub 2020 Jul 16.
Among various in vitro plant culture systems, hairy root systems seem to be one of the most appealing methods of recombinant protein production due to their advantages in combining both whole-plant cultivation and suspension cell culture platform. This is a report on production and secretion of a recombinant pharmaceutically active protein from hairy roots cultures of Withania somnifera to improve the economic potential of this plant for the production pharmaceutical compounds. In this study, we selected and synthesized a codon-optimized globular adiponectin (gAd) gene with a calreticulin signal peptide and cloned the sequence into a plant expression binary vector containing a nptII gene as a selectable marker gene. The transgenic hairy roots were produced by Agrobacterium rhizogenes-mediated transformation protocol developed by our group. Among ten established nptII positive hairy roots lines, six colons significantly accumulated gAd protein in the biomass and extracellular medium. The presence of gAd was confirmed by western blot analysis of root extracts. The maximum level of hairy root biomass, growth rate (GR), intra- and extracellular gAd expressions were obtained after 25-26 days of culture on MS medium. The maximum level of intra- and extracellular gAd proteins were found to be 15.19 μg/gFW and 215.7 μg/L, respectively, which resulted in a significant decrease in the amount of intra- and extracellular withanolide A and withaferin A production. The addition of PVP, KNO3 and NaCl significantly increased the level of extracellular gAd by approximately 13 folds. This improvement could significantly increase the amount of intra- and extracellular withanolide A and withaferin A production, too. The recombinant gAd produced from W. somnifera is functional as proved by induction the phosphorylation of ACC in C2C12 muscle cells, as its functional amount was 5.1-fold more than gAd produced from E. coli and 45 % lower than CHO cells.
在各种体外植物培养系统中，发根系统似乎是生产重组蛋白最有吸引力的方法之一，因为它结合了整株植物培养和悬浮细胞培养平台的优势。这是一份关于利用印度人参发根培养物生产和分泌重组药用活性蛋白的报告，旨在提高该植物生产药物化合物的经济潜力。在这项研究中，我们选择并合成了一个密码子优化的球状脂联素（gAd）基因，带有钙网蛋白信号肽，并将该序列克隆到含有 nptII 基因作为选择标记基因的植物表达二元载体中。转基因发根是通过我们小组开发的根瘤农杆菌介导的转化方案产生的。在十个建立的 nptII 阳性发根系中，有六个系在生物量和细胞外培养基中显著积累了 gAd 蛋白。通过对根提取物的 Western blot 分析证实了 gAd 的存在。在 MS 培养基上培养 25-26 天后，获得了最大的发根生物量、生长率（GR）、细胞内和细胞外 gAd 表达水平。细胞内和细胞外 gAd 蛋白的最大水平分别为 15.19μg/gFW 和 215.7μg/L，这导致细胞内和细胞外 withanolide A 和 withaferin A 产量显著下降。添加 PVP、KNO3 和 NaCl 可使细胞外 gAd 水平显著增加约 13 倍。这种改进也可以显著增加细胞内和细胞外 withanolide A 和 withaferin A 的产量。从 W. somnifera 中产生的重组 gAd 是有功能的，这一点通过诱导 C2C12 肌肉细胞中 ACC 的磷酸化得到证明，其功能量比大肠杆菌产生的 gAd 高 5.1 倍，比 CHO 细胞低 45%。
