Department of Biotechnology, Institute of Science, High Technology and Environmental Science, Graduate University of Advanced Technology, P.O. Box 76315-117, Kerman, Iran.
Department of Plant Breeding, Faculty of Agiculture, Tarbiat Modares University, P.O. Box 14115-336, Tehran, Iran.
Plant Physiol Biochem. 2016 Nov;108:139-144. doi: 10.1016/j.plaphy.2016.06.029. Epub 2016 Jun 25.
Plants are low cost platforms for the production of recombinant proteins, but their complexity renders the purification of plant recombinant proteins more difficult than proteins expressed in yeast or bacteria. Plastid transformation enables high-level expression of foreign genes and the accumulation of recombinant proteins in plastid organelles. Histidine (His) tags are widely used for affinity purification of recombinant proteins in a nickel column. The human tissue-type plasminogen activator (tPA) is one of the most important pharmaceutical recombinant proteins involved in the breakdown of blood clots in different parts of the body. The truncated form of the tissue plasminogen activator (K2S) has a longer plasma half-life, better diffusion into the clot, and higher fibrinolytic activity. In a construct designed to insert the K2S gene in the tobacco chloroplast, the sequence of six histidines and a factor Xa protease site was fused to the C-terminus of the K2S protein. The presence and amount of tPA recombinant protein in transplastomic tobacco plants was estimated by ELISA analysis using a specific antibody. The protein was purified from total soluble protein, insoluble protein aggregates and the protein was extracted from the isolated chloroplast using nickel resin and a chromatography column. After digestion of the purified protein with factor Xa, the presence of the purified tPA protein was confirmed by western blot analysis.
植物是生产重组蛋白的低成本平台，但由于其复杂性，使得植物重组蛋白的纯化比在酵母或细菌中表达的蛋白更困难。质体转化使外源基因的高水平表达和重组蛋白在质体细胞器中的积累成为可能。组氨酸（His）标签广泛用于镍柱亲和纯化重组蛋白。人组织型纤溶酶原激活剂（tPA）是参与体内不同部位血栓溶解的最重要的药物重组蛋白之一。组织纤溶酶原激活剂（K2S）的截断形式具有更长的血浆半衰期、更好的向血栓扩散和更高的纤维蛋白溶解活性。在设计用于将 K2S 基因插入烟草叶绿体的构建体中，将 6 个组氨酸序列和因子 Xa 蛋白酶位点融合到 K2S 蛋白的 C 末端。通过使用特异性抗体的 ELISA 分析来估计转基因烟草植物中转录体中 tPA 重组蛋白的存在和含量。该蛋白从总可溶性蛋白、不溶性蛋白聚集体中纯化，并使用镍树脂和色谱柱从分离的叶绿体中提取。用因子 Xa 消化纯化的蛋白后，通过 Western blot 分析确认了纯化的 tPA 蛋白的存在。
