Recombinant protein condensation inside E. coli enables the development of building blocks for bioinspired materials engineering – Biomimetic spider silk protein as a case study. (15th December 2022)

• Record Type: Journal Article
• Authors: Gabryelczyk, Bartosz; Sammalisto, Fred-Eric; Gandier, Julie-Anne; Feng, Jianhui; Beaune, Grégory; Timonen, Jaakko V.I.; Linder, Markus B.
• Is Part Of: Materials today bio. Volume 17(2023)
• Publication Date: 2022-12-15
• Subjects: Liquid-liquid phase separation -- Protein condensation -- Intracellular condensate -- Spider silk -- Protein expression in E. coli -- Protein-based material; Materials science -- Periodicals; Biomedical engineering -- Periodicals; Biomedical materials -- Periodicals; 620.1
• DOI: 10.1016/j.mtbio.2022.100492
• Languages: English
• ISSNs: 2590-0064

Abstract

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. Graphical abstract: Image 1 Highlights: The silk protein NT2RepCT undergoes liquid-liquid phase separation in E. coli cells. Experiments showed liquid-like properties of the resulting protein condensates. The ability to undergo condensation is suggested to link to high production yields. Final fiber formation is linked to the condensation of NT2RepCT. The general strategy of cellular condensation for protein selection is suggested.