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

本项目的目的是提高转基因植物悬浮培养物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）和白细胞介素（IL-2、IL-4和IL-10）被用作模型分泌的转基因蛋白。哺乳动物蛋白在植物细胞中的表达将通过使用强组成型和诱导型启动子，或通过添加烟草蚀纹病毒（TEV）的非翻译前导序列来增加。将通过添加各种植物信号肽序列来检查转基因蛋白的靶向和分泌。研究糖基化对内质网（ER）靶向/滞留序列的影响。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，将使蛋白质回收率最大化。一种新的亲和层析生物反应器是要开发的，使用镍螯合物氨三乙酸柱，以增加在原位生产和分离的分泌的哺乳动物蛋白质从植物悬浮培养。

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.