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Expanding the genetic code of Saccharomyces cerevisiae with methionine analogues
Author(s) -
Wiltschi Birgit,
Wenger Waltraud,
Nehring Sebastian,
Budisa Nediljko
Publication year - 2008
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.1632
Subject(s) - methionine , saccharomyces cerevisiae , biology , yeast , biochemistry , amino acid , auxotrophy , norleucine , gene , escherichia coli
We replaced the single N‐terminal methionine in heterologously expressed human Cu/Zn superoxide dismutase with the non‐canonical methionine analogues homopropargylglycine and norleucine in the yeast Saccharomyces cerevisiae . Our non‐canonical amino acid incorporation protocol involves a two‐step procedure. In the first step, the methionine auxotrophic yeast cells are accumulated in synthetic medium containing methionine while the target protein production is shut off. After a short methionine depletion phase, the cells are transferred to inducing medium that contains the methionine analogue instead of methionine and target protein expression is switched on. The initially low level incorporation of ∼12% could be elevated to 40% by increasing the non‐canonical amino acid concentration in the medium by 10‐fold. With this approach we were able to produce up to 5 mg substituted protein per litre of yeast culture. Copyright © 2008 John Wiley & Sons, Ltd.