Department of Plant Breeding & Biotechnology, Faculty of Agriculture, University of Tabriz, 51666, Tabriz, Iran.
Department of Animal Biology, Faculty of Natural Science, University of Tabriz, 51666, Tabriz, Iran.
Biol Res. 2022 Oct 23;55(1):32. doi: 10.1186/s40659-022-00400-7.
Designed mimetic molecules are attractive tools in biopharmaceuticals and synthetic biology. They require mass and functional production for the assessment of upcoming challenges in the near future. The DARPin family is considered a mimetic pharmaceutical peptide group with high affinity binding to specific targets. DARPin G3 is designed to bind to the HER2 (human epidermal growth factor receptor 2) tyrosine kinase receptor. Overexpression of HER2 is common in some cancers, including breast cancer, and can be used as a prognostic and predictive tool for cancer. The chloroplasts are cost-effective alternatives, equal to, and sometimes better than, bacterial, yeast, or mammalian expression systems. This research examined the possibility of the production of the first antibody mimetic, DARPin G3, in tobacco chloroplasts for HER2 imaging in oncology.
The chloroplast specific DARPin G3 expression cassette was constructed and transformed into N. tabacum chloroplasts. PCR and Southern blot analysis confirmed integration of transgenes as well as chloroplastic and cellular homoplasmy. The Western blot analysis and ELISA confirmed the production of DARPin G3 at the commercial scale and high dose with the rate of 20.2% in leaf TSP and 33.7% in chloroplast TSP. The functional analysis by ELISA confirmed the binding of IMAC purified chloroplast-made DARPin G3 to the extracellular domain of the HER2 receptor with highly effective picomolar affinities. The carcinoma cellular studies by flow cytometry and immunofluorescence microscopy confirmed the correct functioning by the specific binding of the chloroplast-made DARPin G3 to the HER2 receptor on the surface of HER2-positive cancer cell lines.
The efficient functional bioactive production of DARPin G3 in chloroplasts led us to introduce plant chloroplasts as the site of efficient production of the first antibody mimetic molecules. This report, as the first case of the cost-effective production of mimetic molecules, enables researchers in pharmaceuticals, synthetic biology, and bio-molecular engineering to develop tool boxes by producing new molecular substitutes for diverse purposes.
设计模拟分子是生物制药和合成生物学中极具吸引力的工具。为了应对近期即将出现的挑战，它们需要大规模和功能性生产。DARPin 家族被认为是一种具有高亲和力结合特定靶标的模拟药物肽组。DARPin G3 被设计用于结合 HER2（人表皮生长因子受体 2）酪氨酸激酶受体。HER2 的过表达在一些癌症中很常见，包括乳腺癌，可作为癌症的预后和预测工具。叶绿体是具有成本效益的替代品，与细菌、酵母或哺乳动物表达系统相当，有时甚至更好。本研究探讨了在烟草叶绿体中生产用于肿瘤学 HER2 成像的首个抗体模拟物 DARPin G3 的可能性。
构建了叶绿体特异性 DARPin G3 表达盒，并转化到 N. tabacum 叶绿体中。PCR 和 Southern blot 分析证实了转基因的整合以及叶绿体和细胞的同型性。Western blot 分析和 ELISA 证实了 DARPin G3 在商业规模和高剂量下的生产，叶片 TSP 中的产量为 20.2%，叶绿体 TSP 中的产量为 33.7%。通过 ELISA 进行的功能分析证实，IMAC 纯化的叶绿体产生的 DARPin G3 与 HER2 受体的细胞外结构域结合具有高度有效的皮摩尔亲和力。通过流式细胞术和免疫荧光显微镜对癌细胞的研究证实，叶绿体产生的 DARPin G3 通过与 HER2 阳性癌细胞系表面的 HER2 受体的特异性结合而正确发挥作用。
DARPin G3 在叶绿体中的高效功能性生物活性生产使我们能够将植物叶绿体作为高效生产首个抗体模拟分子的场所。本报告作为低成本生产模拟分子的首例案例，使药物学、合成生物学和生物分子工程领域的研究人员能够通过生产用于各种目的的新型分子替代品来开发工具包。
