Magnuson N S, Linzmaier P M, Gao J W, Reeves R, An G, Lee J M
Department of Microbiology, Washington State University, Pullman 99164-4233, USA.
Protein Expr Purif. 1996 Mar;7(2):220-8. doi: 10.1006/prep.1996.0030.
Increasing the level of recovery of mammalian proteins secreted by a genetically modified Nicotiana tabacum was explored in suspension culture. As a model protein system, a mouse monoclonal antibody heavy chain gamma (MAb HC) with an antigen specificity for p-azophenylarsonate was used. Consistent with findings for other plant cell suspension culture systems expressing proteins with mammalian leader sequences, the synthesized mouse MAb HC was secreted through the plasma membrane. In addition, the majority of the MAb HC was also secreted through the cell wall into the growth medium. However, efficient recovery of the protein was only possible when the protein stabilizing agent, polyvinylpyrrolidone (PVP) was present in the plant cell growth medium. The presence of PVP increased the recovered concentration of secreted protein 35-fold from 0.010 to 0.36 micrograms protein/ml culture medium. Biological activity of the approximately 50-kDa MAb HC polypeptide was demonstrated by arsonate affinity matrix binding as determined by Western blot analysis. In addition to antigen binding activity, the secreted protein also exhibited reactivity to protein G, a protein which specifically binds mouse IgG. These findings are important because they demonstrate that culture conditions can significantly influence the concentration of a biologically active foreign protein secreted from plant cells into the media of suspension cultures. The ability to increase the efficiency of mammalian protein production in plant suspension culture systems should provide significant advantage over protein production in intact transgenic plants which require cultivation, harvesting, and expensive extraction procedures to obtain nonsecreted foreign proteins.
在悬浮培养中探索提高转基因烟草分泌的哺乳动物蛋白的回收率。作为模型蛋白系统，使用了对对氨基苯胂酸具有抗原特异性的小鼠单克隆抗体重链γ（MAb HC）。与其他表达带有哺乳动物前导序列蛋白的植物细胞悬浮培养系统的研究结果一致，合成的小鼠MAb HC通过质膜分泌。此外，大多数MAb HC也通过细胞壁分泌到生长培养基中。然而，只有当植物细胞生长培养基中存在蛋白质稳定剂聚乙烯吡咯烷酮（PVP）时，才能有效地回收该蛋白。PVP的存在使分泌蛋白的回收浓度从0.010微克蛋白/毫升培养基提高到0.36微克蛋白/毫升，提高了35倍。通过蛋白质印迹分析确定的胂酸盐亲和基质结合证明了约50 kDa的MAb HC多肽的生物活性。除了抗原结合活性外，分泌的蛋白还对蛋白G表现出反应性，蛋白G是一种特异性结合小鼠IgG的蛋白。这些发现很重要，因为它们表明培养条件可以显著影响从植物细胞分泌到悬浮培养基中的生物活性外源蛋白的浓度。提高植物悬浮培养系统中哺乳动物蛋白生产效率的能力应该比完整转基因植物中的蛋白生产具有显著优势，完整转基因植物需要培养、收获以及昂贵的提取程序来获得非分泌的外源蛋白。
