title: Codon Optimizing for Increased Membrane Protein Production: A Minimalist Approach.
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.
authors.affiliation: Department of Biochemistry and Biophysics, Center for Biomembrane Research, Stockholm University, Stockholm, Sweden.
authors.forename: Kiavash
authors.initials: K
authors.lastname: Mirzadeh
authors.affiliation: Department of Biochemistry and Biophysics, Center for Biomembrane Research, Stockholm University, Stockholm, Sweden.
authors.forename: Stephen
authors.initials: S
authors.lastname: Toddo
authors.affiliation: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Lyngby, Denmark.
authors.forename: Morten H H
authors.initials: MH
authors.lastname: Nørholm
authors.affiliation: Department of Biochemistry and Biophysics, Center for Biomembrane Research, Stockholm University, Stockholm, Sweden. ddaley@dbb.su.se.
authors.forename: Daniel O
authors.initials: DO
authors.lastname: Daley
mesh_terms: D003001:Cloning, Molecular; D003062:Codon; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development*; D015870:Gene Expression; D015723:Gene Library; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*
chemical_list: D003062:Codon; D008565:Membrane Proteins
keywords: Codon optimization; Membrane protein; Protein expression; Synonymous codon