Materials Today Bio (Dec 2022)

Recombinant protein condensation inside E. coli enables the development of building blocks for bioinspired materials engineering – Biomimetic spider silk protein as a case study

  • Bartosz Gabryelczyk,
  • Fred-Eric Sammalisto,
  • Julie-Anne Gandier,
  • Jianhui Feng,
  • Grégory Beaune,
  • Jaakko V.I. Timonen,
  • Markus B. Linder

Journal volume & issue
Vol. 17
p. 100492

Abstract

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Recombinant expression of proteins destined to form biological materials often results in poor production yields or loss of their function due to premature aggregation. Recently, liquid-liquid phase separation has been proposed as a mechanism to control protein solubility during expression and accumulation in the cytoplasm. Here, we investigate this process in vivo during the recombinant overexpression of the mimetic spider silk mini-spidroin NT2RepCT in Escherichia coli. The protein forms intracellular liquid-like condensates that shift to a solid-like state triggered by a decrease in their microenvironmental pH. These features are also maintained in the purified sample in vitro both in the presence of a molecular crowding agent mimicking the bacterial intracellular environment, and during a biomimetic extrusion process leading to fiber formation. Overall, we demonstrate that characterization of protein condensates inside E. coli could be used as a basis for selecting proteins for both materials applications and their fundamental structure-function studies.

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