We have made a dramatic improvement of the wheat cell-free protein synthesis system. The first key improvement is the method for preparation of the cell-free extract that is free of inhibitory factors of translation reaction. Additional improvements include a method for preparation of transcription-ready templates by PCR, an expression vector for the cell-free system, and the “bilayer” mode reaction method that is much more efficient than the batch mode method and at the same time easy to be performed by human hands and by liquid handling machines. We review here the history of the development and describe the protocols for the most handy “bilayer” method and a more efficient but complicated methods. Information on many examples and variations of the wheat cell-free protein synthesis methods already published elsewhere is then provided so that the readers can understand the power and potential applications of the methods.
Keywords: Cell-free protein synthesis, wheat, split-primer PCR, automation, pEU
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