Increased production of periplasmic proteins in Escherichia coli by directed evolution of the translation initiation region

Kiavash Mirzadeh, Patrick J. Shilling, Rageia Elfageih, Alister J. Cumming, Huanhuan L. Cui, Maja Rennig, Morten H. H. Nørholm, Daniel O. Daley*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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Background: Recombinant proteins are often engineered with an N-terminal signal peptide, which facilitates their secretion to the oxidising environment of the periplasm (gram-negative bacteria) or the culture supernatant (gram-positive bacteria). A commonly encountered problem is that the signal peptide influences the synthesis and secretion of the recombinant protein in an unpredictable manner. A molecular understanding of this phenomenon is highly sought after, as it could lead to improved methods for producing recombinant proteins in bacterial cell factories.
Results: Herein we demonstrate that signal peptides contribute to an unpredictable translation initiation region. A directed evolution approach that selects a new translation initiation region, whilst leaving the amino acid sequence of the signal peptide unchanged, can increase production levels of secreted recombinant proteins. The approach can increase production of single chain antibody fragments, hormones and other recombinant proteins in the periplasm of E. coliConclusions: The study demonstrates that signal peptide performance is coupled to the efficiency of the translation initiation region.
Original languageEnglish
Article number85
JournalMicrobial Cell Factories
Number of pages12
Publication statusPublished - 2020


  • Protein secretion
  • Signal peptide
  • Recombiant protein expression
  • Bacteria
  • Translation initation
  • Directed evolution


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