Decoupling Protein Production from Cell Growth Enhances the Site-Specific Incorporation of Noncanonical Amino Acids in E. coli.

Meritxell Galindo Casas, Patrick Stargardt, Juergen Mairhofer, Birgit Wiltschi*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


The site-specific incorporation of noncanonical amino acids (ncAAs) into proteins by amber stop codon suppression has become a routine method in academic laboratories. This approach requires an amber suppressor tRNACUA to read the amber codon and an aminoacyl-tRNA synthetase to charge the tRNACUA with the ncAA. However, a major drawback is the low yield of the mutant protein in comparison to the wild type. This effect primarily results from the competition of release factor 1 with the charged suppressor tRNACUA for the amber codon at the A-site of the ribosome. A number of laboratories have attempted to improve the incorporation efficiency of ncAAs with moderate results. We aimed at increasing the efficiency to produce high yields of ncAA-functionalized proteins in a scalable setting for industrial application. To do this, we inserted an ncAA into the enhanced green fluorescent protein and an antibody mimetic molecule using an industrial E. coli strain, which produces recombinant proteins independent of cell growth. The controlled decoupling of recombinant protein production from cell growth considerably increased the incorporation of the ncAA, producing substantially higher protein yields versus the reference E. coli strain BL21(DE3). The target proteins were expressed at high levels, and the ncAA was efficiently incorporated with excellent fidelity while the protein function was preserved.
Original languageEnglish
Pages (from-to)3052–3066
Number of pages15
JournalACS Synthetic Biology
Issue number11
Publication statusPublished - 20 Nov 2020


  • affibody
  • amber suppression
  • BL21(DE3) gp2
  • genetic code expansion
  • growth-decoupled protein production
  • noncanonical amino acid
  • Genetic code expansion
  • Amber suppression
  • Affibody
  • Growth-decoupled protein production
  • Noncanonical amino acid

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology (miscellaneous)
  • Biomedical Engineering


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