Optimized Production and Stabilization of Mammalian Proteins Secreted from Transgenic Plant Suspension Cultures

该项目的目的是提高转基因植物悬浮培养中哺乳动物蛋白的表达、分泌和回收。各种人淋巴因子包括人粒细胞-巨噬细胞集落刺激因子(GM-CSF)和白介素2(IL-2)、IL-4和IL-10将被用作模型分泌的转基因蛋白。植物细胞中哺乳动物蛋白的表达将通过使用强的组成性和诱导性启动子或通过添加烟草蚀刻病毒(TEV)的非翻译前导序列来增加。通过添加不同的植物信号肽序列来检测转基因蛋白的靶向性和分泌性。糖基化对内质网(ER)靶向/保留序列的影响有待研究。通过测试各种添加剂在稳定分泌的转基因蛋白质方面的有效性，将最大限度地提高蛋白质回收率。开发了一种新型的亲和层析生物反应器，它使用镍螯合氮三乙酸柱来提高植物悬浮培养物中哺乳动物分泌蛋白的原位生产和分离。

The objective of this project is to enhance the expression, secretion, and recovery of mammalian proteins from transgenic plant suspension cultures. Various human lymphokines including human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukins (IL-2,IL-4, and IL-10) are to be employed as model secreted transgenic proteins. The expression of mammalian proteins in plant cells will be increased by using strong constitutive and inducible promoters, or by adding the non-translated leader sequence of the tobacco etch virus (TEV). The targeting and secretion of transgenic proteins will be examined by adding various plant signal peptide sequences. The effect of glycosylation on the endoplasmic reticulum (ER) targeting/retention sequences are to be investigated. Protein recovery will be maximized through the testing of a variety of additives for their effectiveness in stabilizing secreted transgenic proteins. A novel affinity chromotography bioreactor is to be developed that uses the nickel-chelate nitrilotriacetic column to increase in situ production and separation of the secreted mammalian proteins from plant suspension cultures.