Construction of an in Vitro Gene Screening System of the E. coli EmrE Transporter Using Liposome Display

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• 作者：Atsuko Uyeda ; Shintaro Nakayama ; Yasuhiko Kato ; Hajime Watanabe ; Tomoaki Matsuura
• 刊名：Analytical Chemistry
• 出版年：2016
• 出版时间：December 20, 2016
• 年：2016
• 卷：88
• 期：24
• 页码：12028-12035
• 全文大小：522K
• ISSN：1520-6882

文摘

Liposome display is a method that enables the directed evolution of membrane proteins in vitro. The method is based on the syntheses of membrane proteins using an in vitro transcription–translation system (IVTT) inside cell-sized phospholipid vesicles from a single copy of template DNA. So far, a large number of membrane proteins have been synthesized by IVTT; however, none of these proteins, except for α-hemolysin, has been tested for use in gene screening with liposome display. Here, using EmrE, a multidrug transporter from Escherichia coli,, as a model protein, we developed an in vitro screening system of the transporter gene based on its function, which was made possible by using liposome display. The screening was performed based on two functions of EmrE: substrate transport activity and membrane integration activity. Starting from a mock gene library prepared by mixing an active and an inactive gene, 10- to 35-fold enrichment of the active genes was obtained, which was in the same range as theoretically calculated values. In addition, starting from a random mutagenized gene library of wild-type EmrE, a gene pool exhibiting ethidium bromide (EtBr) transport activity higher than that of the wild-type was obtained, indicating the validity of the established screening system.