title: Virus-Enabled Optimization and Delivery of the Genetic Machinery for Efficient Unnatural Amino Acid Mutagenesis in Mammalian Cells and Tissues.
abstract: Unnatural amino acid (UAA) mutagenesis of recombinant proteins in live mammalian cells requires coexpression of the mutant target, as well as an engineered tRNA/aminoacyl-tRNA synthetase pair. The ability to readily determine the optimal relative expression levels of these three genetic components for efficient expression of the UAA-modified target is highly desirable, but remains challenging to accomplish. Here we report a facile strategy to achieve this by taking advantage of the efficient gene-delivery by a baculovirus vector, which enables systematic variation of the expression level of each genetic component in a population-wide manner. Insights gained from this study led to the design of an optimal expression system, which can be delivered into mammalian cells by a single baculovirus vector to provide significantly improved UAA incorporation efficiency at a low virus load. Furthermore, this optimized baculovirus vector was shown to enable efficient UAA mutagenesis of proteins expressed in mouse brain tissue.
authors.affiliation: Department of Chemistry, Boston College , 2609 Beacon Street, Chestnut Hill, Massachusetts 02467, United States.
authors.forename: Yunan
authors.initials: Y
authors.lastname: Zheng
authors.affiliation: Department of Neuroscience, Columbia University , 550 West 120th Street, New York, New York 10027, United States.
authors.forename: Tommy L
authors.initials: TL
authors.lastname: Lewis
authors.affiliation: Department of Chemistry, Boston College , 2609 Beacon Street, Chestnut Hill, Massachusetts 02467, United States.
authors.forename: Peter
authors.initials: P
authors.lastname: Igo
authors.affiliation: Department of Neuroscience, Columbia University , 550 West 120th Street, New York, New York 10027, United States.
authors.forename: Franck
authors.initials: F
authors.lastname: Polleux
authors.affiliation: Department of Chemistry, Boston College , 2609 Beacon Street, Chestnut Hill, Massachusetts 02467, United States.
authors.forename: Abhishek
authors.initials: A
authors.lastname: Chatterjee
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001921:Brain / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D016296:Mutagenesis; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D000596:Amino Acids; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D012343:RNA, Transfer
keywords: mammalian genetic code expansion; unnatural amino acid incorporation; viral vector