Institute of Agricultural Biotechnology, Jilin Academy of Agricultural Sciences, Changchun, China.
Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan.
Growth Factors. 2023 Feb;41(1):20-31. doi: 10.1080/08977194.2022.2150187. Epub 2022 Dec 1.
Human epidermal growth factor (hEGF) is an important therapeutic compound with multiple applications particularly in pharmaceutical industry. Human EGF has already been expressed in different expression systems, however, the production of hEGF with bioactivity in chloroplasts has not been successful so far. In this study, we expressed a 6 × His-tagged hEGF in tobacco chloroplasts in its native conformation for the potential of large-scale production of hEGF for industrial applications. Several transplastomic plant lines were obtained, which were screened by PCR (polymerase chain reaction) using primers specific to selectable gene , hEGF- and GFP-coding sequences that were included in the chloroplast expression vector. The selected lines were confirmed to be homoplasmic by PCR verification and Southern blot analysis. Immunoblotting assays of homoplasmic lines using antibodies raised against hEGF confirmed the accumulation of hEGF in transplastomic plants and the ELISA results demonstrated the expression levels of hEGF were between 0.124% and 0.165% of the total soluble proteins (TSP), namely, 23.16-25.77 ng/g of the fresh weight. In terms of activity, the data from cell proliferation and elongation assays showed that the tobacco-derived recombinant hEGF was as bioactive as its commercial counterpart. To our knowledge, this is the first report of recombinant production of hEGF with native bioactivity form in the chloroplast stroma. Overall, our results demonstrate the potential of higher plant chloroplasts for the production of a human therapeutic, hEGF, in an active conformation.
人表皮生长因子（hEGF）是一种重要的治疗化合物，具有多种应用，特别是在制药行业。hEGF 已经在不同的表达系统中表达过，然而，到目前为止，在叶绿体中具有生物活性的 hEGF 的生产尚未成功。在这项研究中，我们以天然构象在烟草叶绿体中表达了一个 6×His 标记的 hEGF，以期大规模生产用于工业应用的 hEGF。获得了几个转基因植物系，这些植物系通过使用针对选择基因、hEGF 和 GFP 编码序列的引物进行 PCR（聚合酶链反应）筛选，这些序列包含在叶绿体表达载体中。通过 PCR 验证和 Southern blot 分析，对选定的系进行了同源性验证。使用针对 hEGF 产生的抗体对同源性系进行的免疫印迹分析证实了 hEGF 在转基因植物中的积累，ELISA 结果表明 hEGF 的表达水平在总可溶性蛋白（TSP）的 0.124%到 0.165%之间，即新鲜重量的 23.16-25.77ng/g。就活性而言，细胞增殖和伸长测定的数据表明，烟草衍生的重组 hEGF 与人用重组 hEGF 具有相同的生物活性。据我们所知，这是第一个在叶绿体基质中以天然生物活性形式重组生产 hEGF 的报告。总的来说，我们的结果表明，高等植物叶绿体具有生产活性构象的人治疗性 hEGF 的潜力。
