Abstract
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 mini-library by PCR; and (3) screening for high-expressing clones.
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Acknowledgments
This work was supported by a grant from the Swedish Research Council to DOD, and by the Novo Nordisk Foundation to MHHN.
Conflict of interest statement: The method considered here has been described in a patent submitted by CloneOpt AB (SE1451553-0). KM, ST, and DOD are founders and shareholders in CloneOpt AB.
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Mirzadeh, K., Toddo, S., Nørholm, M.H.H., Daley, D.O. (2016). Codon Optimizing for Increased Membrane Protein Production: A Minimalist Approach. In: Mus-Veteau, I. (eds) Heterologous Expression of Membrane Proteins. Methods in Molecular Biology, vol 1432. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3637-3_4
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DOI: https://doi.org/10.1007/978-1-4939-3637-3_4
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