Zhao Wei, Zhou Lu-Yang, Kong Jing, Huang Ze-Hao, Gao Ya-Di, Zhang Zhong-Xia, Zhou Yong-Jie, Wu Ruo-Yu, Xu Hong-Jun, An Sheng-Jun
Hebei Provincial Engineering Laboratory of Plant Bioreactor Preparation Technology, Hebei University of Chinese Medicine, No. 326 Xinshi South Road, Shijiazhuang, 050090, Hebei, China.
School of Nursing of Hebei University of Chinese Medicine, No. 326 Xinshi South Road, Shijiazhuang, 050090, Hebei, China.
Bioresour Bioprocess. 2023 Jan 21;10(1):4. doi: 10.1186/s40643-023-00623-w.
Apolipoprotein A-I (Apo A-I) is a natural mutant of Apolipoprotein. It is currently the only protein that can clear arterial wall thrombus deposits and promptly alleviate acute myocardial ischemia. Apo A-I is considered as the most promising therapeutic protein for treating atherosclerotic diseases without obvious toxic or side effects. However, the current biopharmaceutical platforms are not efficient for developing Apo A-I. The objectives of this research were to express Apo A-I using the genetic transformation ability of N. tabacum. The method is to clone the coding sequence of Apo A-I into the plant binary expression vector pCHF3 with a Flag/His6/GFP tag. The constructed plasmid was transformed into N. tabacum by a modified agrobacterium-mediated method, and transformants were selected under antibiotic stress. PCR, RT-qPCR, western blot and co-localization analysis was used to further verify the resistant N. tabacum. The stable expression and transient expression of N. tabacum were established, and the pure product of Apo A-I was obtained through protein A/G agarose. The results showed that Apo A-I was expressed in N. tabacum with a yield of 0.05 mg/g leaf weight and the purity was 90.58% ± 1.65. The obtained Apo A-I protein was subjected to amino acid sequencing. Compared with the theoretical sequence of Apo A-I, the amino acid coverage was 86%, it is also found that Cysteine replaces Arginine at position 173, which indicates that Apo A-I, a mutant of Apo A-I, is accurately expressed in N. tabacum. The purified Apo A-I protein had a lipid binding activity. The established genetic modification N. tabacum will provide a cost-effective system for the production of Apo A-I. Regarding the rapid propagation of N. tabacum, this system provides the possibility of large-scale production and accelerated clinical translation of Apo A-I.
载脂蛋白A-I（Apo A-I）是载脂蛋白的一种天然突变体。它是目前唯一能够清除动脉壁血栓沉积物并迅速缓解急性心肌缺血的蛋白质。Apo A-I被认为是治疗动脉粥样硬化疾病最有前景的治疗性蛋白质，且无明显毒副作用。然而，当前的生物制药平台在开发Apo A-I方面效率不高。本研究的目的是利用烟草的遗传转化能力来表达Apo A-I。方法是将Apo A-I的编码序列克隆到带有Flag/His6/GFP标签的植物双元表达载体pCHF3中。通过改良的农杆菌介导法将构建好的质粒转化到烟草中，并在抗生素胁迫下筛选转化体。使用PCR、RT-qPCR、蛋白质免疫印迹和共定位分析进一步验证抗性烟草。建立了烟草的稳定表达和瞬时表达，并通过蛋白A/G琼脂糖获得了Apo A-I的纯品。结果表明，Apo A-I在烟草中表达，产量为0.05 mg/g叶重，纯度为90.58%±1.65。对获得的Apo A-I蛋白进行氨基酸测序。与Apo A-I的理论序列相比，氨基酸覆盖率为86%，还发现第173位的半胱氨酸取代了精氨酸，这表明Apo A-I的突变体Apo A-I在烟草中得到了准确表达。纯化的Apo A-I蛋白具有脂质结合活性。所建立的转基因烟草将为Apo A-I的生产提供一个具有成本效益的系统。关于烟草的快速繁殖，该系统为Apo A-I的大规模生产和加速临床转化提供了可能性。
