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Simultaneous in vitro protein synthesis using solid-phase DNA template.

Mary Kate W DiTursi·Junhoe Cha·Matthew R Newman·Jonathan S Dordick
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Abstract

In vitro protein synthesis is rapidly becoming an accepted tool in functional genomic analysis. We have demonstrated the in vitro synthesis of firefly luciferase on solid-phase template DNA, bound to wells in 96-well plates, using simultaneous transcription and translation in a wheat-germ extract system. The bound DNA template was stable and did not release during transcription. Coupled translation resulted in ca. 1.2 ng/microL luciferase synthesized, which is ca. one-fifth of that synthesized using conventional solution-phase coupled transcription and translation. Reuse of the DNA template was influenced by the complexity of the wheat-germ extract, which resulted in fouling of the transcription surface and reduction of protein synthesis after extended use. The approach developed in this study may enable the development of high-throughput, microscale protein synthesis platforms for use in functional genomic analysis.

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Cell-Free SystemDNALuciferasesPhase TransitionTemplates, GeneticTriticumJournal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, Non-P.H.S.

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Mary Kate W DiTursi, Junhoe Cha, Matthew R Newman, Jonathan S Dordick. Simultaneous in vitro protein synthesis using solid-phase DNA template.. https://doi.org/10.1021/bp049772f

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