Periplasmic expression in and release of Fab fragments from Escherichia coli using stress minimisation

Chia-Chang Hsu, Owen Thomas, Timothy Overton

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13 Citations (Scopus)
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BACKGROUND – The bacterium Escherichia coli is a commonly used host for the production of recombinant protein biopharmaceutical products. One class of such molecules is antibody fragments, typified by the Crohn’s disease and rheumatoid arthritis therapy Certolizumab pegol (Cimzia®). Antibody fragments generated in E. coli are often directed to the periplasm, so that disulphide bonding can occur and release can be simplified. However, many recombinant protein products are prone to misfolding and mislocalisation. Here, we optimised the production of a Fab fragment, D1.3, and its release from the periplasm of E. coli using osmotic shock. RESULTS – By minimising stress imposed on the bacterial hosts and monitoring Fab, total protein and DNA concentrations of fractions isolated following osmotic release, we successfully targeted the majority of recombinant Fab to the periplasm and were able to rapidly define optimal harvest points. Coupled optimisation of fermentation and release increased the Fab concentration of the periplasmic extract by more than 20-fold. CONCLUSION – Simultaneous optimisation of fermentation and periplasmic release allowed rapid definition of operational space and generation recombinant protein in a form compatible with downstream processing steps. This methodology could be used for optimisation of the production of a range of periplasmically-targeted recombinant proteins.
Original languageEnglish
Pages (from-to)815-822
JournalJournal of Chemical Technology and Biotechnology
Issue number3
Early online date26 Feb 2015
Publication statusPublished - 21 Mar 2016


  • recombinant protein production
  • Fab fragment
  • periplasmic release
  • high cell density culture


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