Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang, Korea.
Department of Life Science, Pohang University of Science and Technology, Pohang, Korea.
Plant Biotechnol J. 2019 Jun;17(6):1094-1105. doi: 10.1111/pbi.13040. Epub 2018 Dec 8.
Plants have recently received a great deal of attention as a means of producing recombinant proteins. Despite this, a limited number of recombinant proteins are currently on the market and, if plants are to be more widely used, a cost-effective and efficient purification method is urgently needed. Although affinity tags are convenient tools for protein purification, the presence of a tag on the recombinant protein is undesirable for many applications. A cost-effective method of purification using an affinity tag and the removal of the tag after purification has been developed. The family 3 cellulose-binding domain (CBM3), which binds to microcrystalline cellulose, served as the affinity tag and the small ubiquitin-related modifier (SUMO) and SUMO-specific protease were used to remove it. This method, together with size-exclusion chromatography, enabled purification of human interleukin-6 (hIL6) with a yield of 18.49 mg/kg fresh weight from leaf extracts of Nicotiana benthamiana following Agrobacterium-mediated transient expression. Plant-produced hIL6 (P-hIL6) contained less than 0.2 EU/μg (0.02 ng/mL) endotoxin. P-hIL6 activated the Janus kinase-signal transducer and activator of transcriptional pathways in human LNCaP cells, and induced expression of IL-21 in activated mouse CD4 T cells. This approach is thus a powerful method for producing recombinant proteins in plants.
植物作为生产重组蛋白的一种手段，近来受到了广泛关注。尽管如此，目前市面上仅有少数几种重组蛋白，而且如果要更广泛地应用植物，就迫切需要一种经济高效的纯化方法。虽然亲和标签是用于蛋白质纯化的便利工具，但对于许多应用来说，在重组蛋白上存在标签是不理想的。本文开发了一种使用亲和标签进行经济高效的纯化，并在纯化后去除标签的方法。纤维素结合域家族 3（CBM3）作为亲和标签，与微晶纤维素结合，并用小泛素相关修饰物（SUMO）和 SUMO 特异性蛋白酶将其去除。该方法与排阻层析相结合，能够从经农杆菌介导的瞬时表达的 Nicotiana benthamiana 叶片提取物中，以每公斤鲜重 18.49 毫克的得率纯化人白细胞介素 6（hIL6）。植物生产的 hIL6（P-hIL6）中的内毒素含量低于 0.2EU/μg（0.02ng/mL）。P-hIL6 激活了人 LNCaP 细胞中的 Janus 激酶信号转导和转录激活因子途径，并诱导激活的小鼠 CD4 T 细胞中表达 IL-21。因此，该方法是在植物中生产重组蛋白的一种有效方法。
