- Gabriel Ma, Biomedical Engineering, University of Delaware
- Mark Blenner, Chemical Engineering, University of Delaware
Recombinant protein synthesis is an important technique in producing highly-efficient specific activities of proteins. Chinese hamster ovary (CHO) cells are an epithelial cell line derived from the ovary of the Chinese hamster, to enable improved recombinant protein synthesis. For example, these cells have the ability to produce complicated pharmaceuticals, therapeutic proteins, and have other beneficial properties for laboratory work to withstand various pH and temperature changes and ideal post-translational modifications. While it is in high demand for difficult secretory recombinant biotherapeutics, there needs to be more research focusing on the beneficial role of lipids in CHO cells and exploring the use of polyunsaturated fatty acids (PUFAs) to simplify media formulation, resulting in healthier cell lines and less variable media. Additionally, the study investigates the upregulation of monounsaturated fatty acid (MUFA) production to enlarge the endoplasmic reticulum, leading to improved protein productivity and reduced ER stress, addressing the high demand for difficult secretory recombinant biotherapeutics. Lipid metabolism was performed through the usage of n-3 and n-6 desaturase enzymes. The GoldenGate Expression system then was used to ligate multiple genes into the expression vector, to allow simultaneous and directional assembly of multiple DNA fragments into a single piece. The two enzymes were then incorporated in vitro into physically transfected CHO cells through electroporation. We hypothesize that since the enzymes are already present in the system, the overexpressed monounsaturated fatty acid (MUFA) pathway causes polyunsaturated fatty acids (PUFAs) to be overexpressed, improving protein synthesis. In the future, by using cold-shock promoters, we intend to develop a mechanism for lipid synthesis and stress response in the endoplasmic reticulum (ER). Genetically manipulating a CHO cell’s lipid metabolism can improve the cells’ capacity to create a variety of secretory recombinant protein products. Resulting in greater ER size, productivity, and stress response, as well as conditional expression of mRNA.