Preparation method forEscherichia coli S30 extracts completely dependent upon tRNA addition to catalyze cell-free protein synthesis
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A simple method for depletingE. coli S30 extracts of endogenous tRNA has been developed. An ethanolamine-Sepharose® column equilibrated with water selectively captured the tRNA molecules inE. coli S30 extracts. As a result, S30 extracts filtered through this column became completely dependent upon the addition of exogenous tRNA to mediate cell-free protein synthesis reactions. We anticipate that the procedures developed and described will be particularly useful forin vitro suppression reaction studies designed to introduce unnatural amino acids into protein molecules.
Keywordscell-free protein synthesis unnatural amino acid in vitro suppression tRNA chloramphenicol acetyltransferase erythropoietin
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