Journal
CHEMISTRY-A EUROPEAN JOURNAL
Volume 19, Issue 21, Pages 6824-6830Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/chem.201203923
Keywords
amino acids; cell-free synthesis; enzyme catalysis; protecting groups; protein expression
Categories
Funding
- CSIRO Emerging Science Initiative for Synthetic Enzymes
- ARC Centre of Excellence for Free Radical Chemistry and Biotechnology
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The S30 extract from E. coli BL21 Star (DE3) used for cell-free protein synthesis removes a wide range of -amino acid protecting groups by cleaving -carboxyl hydrazides; methyl, benzyl, tert-butyl, and adamantyl esters; tert-butyl and adamantyl carboxamides; -amino form-, acet-, trifluoroacet-, and benzamides; and side-chain hydrazides and esters. The free amino acids are produced and incorporated into a protein under standard conditions. This approach allows the deprotection of amino acids to be carried out in situ to avoid separate processing steps. The advantages of this approach are demonstrated by the efficient incorporation of the chemically intractable (S)-4-fluoroleucine, (S)-4,5-dehydroleucine, and (2S,3R)-4-chlorovaline into a protein through the direct use of their respective precursors, namely, (S)-4-fluoroleucine hydrazide, (S)-4,5-dehydroleucine hydrazide, and (2S,3R)-4-chlorovaline methyl ester. These results also show that the fluoro- and dehydroleucine and the chlorovaline are incorporated into a protein by the normal biosynthetic machinery as substitutes for leucine and isoleucine, respectively.
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