Fischer R, Liao Y C, Drossard J
Institut für Biologie I (Botanik)/Molekulargenetik), RWTH Aachen, Germany.
J Immunol Methods. 1999 Jun 24;226(1-2):1-10. doi: 10.1016/s0022-1759(99)00058-7.
A TMV-specific full-size murine IgG-2b/K antibody (mAb24) was expressed in a Nicotiana tabacum cv. Petite Havana SR1 suspension culture (P9s), which was derived from a stably transformed transgenic plant (P9). The integration of an N-terminal murine leader peptide directed the assembled immunoglobulin for secretion. However, in suspension culture, the full-size recombinant antibody, rAb24, was retained by the plant cell wall and was not present in the culture medium. rAb24 expression reached a basal level of 15 microg per gram wet cell weight, corresponding to 0.3% of the total soluble plant cell protein. The level of rAb24 could be increased three-fold by amino acid supplementation of the culture medium. For purification of the recombinant antibody from batch-cultured tobacco suspension cells, the primary plant cell wall was partially digested by enzymatic treatment. This resulted in a total release of recombinant full-size rAb24 into the extraction buffer. A three-step procedure was used to purify the immunoglobulins, starting with cross-flow filtration (step 1) followed by protein A affinity chromatography (step 2) and gel filtration as a final purification step (step 3). This procedure gave a recovery of more than 80% of the expressed rAb24 from plant cell extracts. SDS-PAGE, IEF and immunoblot analyses demonstrated a high degree of homogeneity for the affinity-purified rAb24. An ELISA procedure demonstrated that the specificity and affinity of the protein A affinity purified antibody was indistinguishable from its murine counterpart, indicating the potential of plant cell suspension cultures as bio-reactors for the production of recombinant antibodies.
一种烟草花叶病毒（TMV）特异性的全尺寸鼠源IgG-2b/K抗体（单克隆抗体24，mAb24）在烟草品种 Petit Havana SR1 的悬浮培养物（P9s）中表达，该悬浮培养物源自稳定转化的转基因植物（P9）。N 端鼠源前导肽的整合引导组装好的免疫球蛋白进行分泌。然而，在悬浮培养中，全尺寸重组抗体 rAb24 被植物细胞壁保留，未出现在培养基中。rAb24 的表达达到每克湿细胞重 15 微克的基础水平，相当于植物细胞总可溶性蛋白的 0.3%。通过在培养基中补充氨基酸，rAb24 的水平可提高三倍。为了从分批培养的烟草悬浮细胞中纯化重组抗体，通过酶处理部分消化了植物初级细胞壁。这导致重组全尺寸 rAb24 完全释放到提取缓冲液中。采用三步法纯化免疫球蛋白，首先是错流过滤（步骤 1），接着是蛋白 A 亲和层析（步骤 2），最后一步是凝胶过滤作为最终纯化步骤（步骤 三）。该方法从植物细胞提取物中回收了超过 80%表达的 rAb24。SDS-PAGE、IEF 和免疫印迹分析表明亲和纯化的 rAb24 具有高度同质性。ELISA 程序表明蛋白 A 亲和纯化抗体的特异性和亲和力与其鼠源对应物无法区分，这表明植物细胞悬浮培养物作为生产重组抗体的生物反应器具有潜力。
