Simultaneous in vitro protein synthesis using solid-phase DNA template.
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.
DiTursi
Mary Kate W
MK
Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180, USA.
Cha
Junhoe
J
Newman
Matthew R
MR
Dordick
Jonathan S
JS
D002474:Cell-Free System; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D008156:Luciferases / Q000096:biosynthesis* / Q000138:chemical synthesis*; D044367:Phase Transition; D013698:Templates, Genetic; D014908:Triticum / Q000737:chemistry*
D004247:DNA; D008156:Luciferases