Reengineering a gene with synonymous codons is a popular approach for increasing production levels of recombinant proteins. Here we present a minimalist alternative to this method, which samples synonymous codons only at the second and third positions rather than the entire coding sequence. As demonstrated with two membrane-embedded transporters in Escherichia coli, the method was more effective than optimizing the entire coding sequence. The method we present is PCR based and requires three simple steps: (1) the design of two PCR primers, one of which is degenerate; (2) the amplification of a minilibrary by PCR; and (3) screening for high-expressing clones.
CITATION STYLE
Mirzadeh, K., Toddo, S., Nørholm, M. H. H., & Daley, D. O. (2016). Codon optimizing for increased membrane protein production: A minimalist approach. In Methods in Molecular Biology (Vol. 1432, pp. 53–61). Humana Press Inc. https://doi.org/10.1007/978-1-4939-3637-3_4
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