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

doi:10.1016/j.mtbio.2022.100492

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

Mater Today Bio. 2022 Nov 14:17:100492. doi: 10.1016/j.mtbio.2022.100492. eCollection 2022 Dec 15.

Authors

Bartosz Gabryelczyk¹, Fred-Eric Sammalisto¹, Julie-Anne Gandier¹, Jianhui Feng¹, Grégory Beaune², Jaakko V I Timonen², Markus B Linder¹

Affiliations

¹ Department of Bioproducts and Biosystems, School of Chemical Engineering, Aalto University, Espoo, Finland.
² Department of Applied Physics, School of Science, Aalto University, Espoo, Finland.

Abstract

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.

Keywords: Intracellular condensate; Liquid-liquid phase separation; Protein condensation; Protein expression in E. coli; Protein-based material; Spider silk.