Codon Optimizing for Increased Membrane Protein Production: A Minimalist Approach

  • Kiavash Mirzadeh
  • Stephen Toddo
  • Morten H. H. Nørholm
  • Daniel O. Daley
Part of the Methods in Molecular Biology book series (MIMB, volume 1432)


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.

Key words

Membrane protein Protein expression Codon optimization Synonymous codon 



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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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Kiavash Mirzadeh
    • 1
  • Stephen Toddo
    • 1
  • Morten H. H. Nørholm
    • 2
  • Daniel O. Daley
    • 1
  1. 1.Department of Biochemistry and Biophysics, Center for Biomembrane ResearchStockholm UniversityStockholmSweden
  2. 2.Novo Nordisk Foundation Center for BiosustainabilityTechnical University of DenmarkLyngbyDenmark

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