Hein M B, Tang Y, McLeod D A, Janda K D, Hiatt A
Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 92037.
Biotechnol Prog. 1991 Sep-Oct;7(5):455-61. doi: 10.1021/bp00011a011.
Expression of cDNA constructs encoding full-length mouse immunoglobulin chains with their native leader sequences or fusion constructs substituting the native leader with a pre-pro sequence derived from Saccharomyces cerevisiae yielded blocked N-termini on the gamma chain or the correct amino terminal sequence on the mature kappa chain. Lectin binding assays revealed that assembled immunoglobulin complexes contained a glycosylated heavy chain. The attached glycan was resistant to digestion by endoglycosidase H and its lectin binding pattern was distinguishable from that of the mammalian glycan. The results indicated processing of the immunoglobulin carbohydrate in the tobacco Golgi to yield a complex oligosaccharide. Secretion of antibody by protoplasts isolated from regenerated transgenic plants or from suspension callus cells was demonstrated by pulse-chase labeling experiments. When purified, the tobacco-produced antibody was found to possess the antigen binding and catalytic properties of the murine monoclonal antibody. Kinetic parameters (Km, Ki, Vmax, and kcat) of the tobacco-derived antibody were comparable to those of the mouse-derived antibody. The results in general show that the endomembrane system of tobacco cells possesses cognate mechanisms for the recognition of diverse leader sequences. These signals can be used to initiate the assembly, processing, and secretion by plant cells of complex foreign proteins.
编码带有天然前导序列的全长小鼠免疫球蛋白链的cDNA构建体，或用源自酿酒酵母的前原序列替代天然前导序列的融合构建体的表达，在γ链上产生了封闭的N端，而在成熟的κ链上产生了正确的氨基末端序列。凝集素结合试验表明，组装的免疫球蛋白复合物包含一条糖基化的重链。附着的聚糖对内切糖苷酶H的消化具有抗性，其凝集素结合模式与哺乳动物聚糖的不同。结果表明，烟草高尔基体中免疫球蛋白碳水化合物的加工产生了一种复合寡糖。脉冲追踪标记实验证明了从再生转基因植物或悬浮愈伤组织细胞中分离出的原生质体分泌抗体。纯化后发现，烟草产生的抗体具有鼠单克隆抗体的抗原结合和催化特性。烟草来源抗体的动力学参数（Km、Ki、Vmax和kcat）与小鼠来源抗体的相当。总体结果表明，烟草细胞的内膜系统具有识别多种前导序列的同源机制。这些信号可用于启动植物细胞对复杂外源蛋白的组装、加工和分泌。
