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

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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.