Simultaneous in Vitro Protein Synthesis Using Solid-Phase DNA Template

• Published in: Biotechnology progress 2004-11, Vol.20 (6), p.1705-1709
• Main Authors: DiTursi, Mary Kate W., Cha, Junhoe, Newman, Matthew R., Dordick, Jonathan S.
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Biotechnology; Cell-Free System; DNA; DNA - chemistry; DNA - metabolism; Fundamental and applied biological sciences. Psychology; Luciferases - biosynthesis; Luciferases - chemical synthesis; Phase Transition; Protein biosynthesis; Q1; Solid phase methods; Templates; Templates, Genetic; Transcription; Translation; Triticum - chemistry
• ISSN: 8756-7938; 1520-6033
• DOI: 10.1021/bp049772f

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/μL 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.