Virus-Enabled Optimization and Delivery of the Genetic Machinery for Efficient Unnatural Amino Acid Mutagenesis in Mammalian Cells and Tissues

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• 作者：Yunan Zheng ; Tommy L. Lewis Jr. ; Peter Igo ; Franck Polleux ; Abhishek Chatterjee
• 刊名：ACS Synthetic Biology
• 出版年：2017
• 出版时间：January 20, 2017
• 年：2017
• 卷：6
• 期：1
• 页码：13-18
• 全文大小：370K
• ISSN：2161-5063

文摘

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.