Investigation of selective release of periplasmic proteins through pore size analysis and single-cell microscopy in Escherichia coli

Subbarayalu Ramalakshmi, Ramakrishnan Nagasundara Ramanan, Shanmugavel Madhavan, Chien Wei Ooi, Catherine Ching Han Chang, Ian S. Harper, David M. Lewis, Andrew K. Lee, Lizhong He, Arivudainambi Seenichamy

Research output: Contribution to journalArticleResearchpeer-review

Abstract

While fermentation conditions are known to affect cell membrane permeability, the physical impact of osmotic shock as a process to recover periplasmic proteins remains unknown. Here, we demonstrate the impact of alterations in fermentation conditions and osmotic shock on membrane properties, which in turn affect the selective release of specific periplasmic proteins. Our results suggest that the harvesting phase and the temperature of fermentation significantly affect the Young's modulus and the initial pore size of cells, respectively. For cells with a Young's modulus of less than 1 MPa, both the initial pore size and soluble protein expression affect the selective protein release during typical osmotic shock performed using 20 % sucrose. Further increasing the sucrose concentration to 30 % reduces the dependency of the selective protein release on initial pore size. Our findings will assist further in developing better bioprocess conditions to recover periplasmic proteins.

Original languageEnglish
Article number108009
Number of pages12
JournalBiochemical Engineering Journal
Volume171
DOIs
Publication statusPublished - Jul 2021

Keywords

  • Atomic force microscopy
  • Fluorescence microscopy osmotic shock
  • interferon-α2b
  • Process optimization
  • Single-cell

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