Site-specific incorporation of unnatural amino acids into receptors expressed in mammalian cells

mammalian cell;unnatural amino acids

Monahan, Sarah Lynn ; Dougherty, Dennis A.

University:California Institute of Technology

Department:Chemistry and Chemical Engineering

关键词: mammalian cell;    unnatural amino acids;   

Others  :  https://thesis.library.caltech.edu/2046/1/SLMChapter1.pdf

美国|英语

来源: Caltech THESIS

【 摘 要 】

Unnatural amino acid incorporation into proteins by nonsense suppression has proven to be a valuable tool for structure-function studies.Using the in vivo nonsense suppression methodology, information on ligand binding and ion channel gating mechanisms has been obtained on a variety of ion channels.To date, such studies have been limited to the Xenopus oocyte heterologous expression system.There would be clear benefits to expanding the technology to a mammalian cell expression system.This would provide a more relevant environment for many proteins of mammalian origin and would allow for studies of cell-specific signal transduction pathways.We describe here unnatural amino acid incorporation into channels and receptors expressed in mammalian cells.Presented is a general method to deliver mRNA or DNA that codes for a protein of interest, amber suppressor tRNA, and a reporter gene to mammalian cells.Chapter 2 describes in detail the screening of several suppressor tRNAs, as well as various transfection methods tested for tRNA delivery including electroporation, lipofection, peptide-mediated transfection and biolistics.It was found that electroporation was the best method to deliver tRNA to adherent cells, and that THG73 was the most efficient suppressor tRNA.Chapter 3 describes studies, aimed at optimizing the protocol, that involved co-electroporation of a human serine amber suppressor tRNA with the DNA or mRNA corresponding to the protein of interest into adherent cells.This leads to highly efficient delivery of these components and efficient nonsense suppression.We demonstrate this for both enhanced green fluorescent protein and nicotinic acetylcholine receptor expression in CHO-K1 cells.We also show that the approach is successful in cultured hippocampal neurons.Finally, Chapter 4 demonstrates the application of the electroporation method to the delivery of aminoacyl-tRNA to cells for unnatural amino acid incorporation into the nicotinic acetylcholine receptor.When chemically aminoacylated with natural or unnatural amino acids, THG73 delivers the amino acid site-specifically into receptors expressed in CHO-K1 cells.Electrophysiology clearly reveals the expected shift in dose-response relations, establishing that the desired unnatural amino acid has been incorporated.In conclusion, we describe the first general method for unnatural amino acid incorporation in mammalian cells.