In vivo liquid–liquid phase separation protects amyloidogenic and aggregation‐prone peptides during overexpression in Escherichia coli

Abstract

Studying pathogenic effects of amyloids requires homogeneous amyloidogenic peptide samples. Recombinant production of these peptides is challenging due to their susceptibility to aggregation and chemical modifications. Thus, chemical synthesis is primarily used to produce amyloidogenic peptides suitable for high‐resolution structural studies. Here, we exploited the shielded environment of protein condensates formed via liquid–liquid phase separation (LLPS) as a protective mechanism against premature aggregation. We designed a fusion protein tag undergoing LLPS in Escherichia coli and linked it to highly amyloidogenic peptides, including β amyloids. We find that the fusion proteins form membraneless organelles during overexpression and remain fluidic‐like. We also developed a facile purification method of functional Aβ peptides free of chromatography steps. The strategy exploiting LLPS can be applied to other amyloidogenic, hydrophobic, and repetitive peptides that are otherwise difficult to produce.

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

“…However, they could not be associated with an insoluble or condensed state exclusively based on these morphological features. In previous studies, both inclusion bodies [ 41 , 42 ] and liquid-like protein condensates [ 14 , 24 , 25 ] have been observed to locate to either one or both poles of the E. coli cell with similar morphologies. The mechanism behind localization of proteins at both poles of the cells is called nucleoid occlusion and is widely observed in E. coli [ 43 ].…”

“…LLPS provides a way to regulate proteins’ transition from a soluble (liquid-like state) into the solid final form of a material. During this process the condensed phase can also serve to prevent premature aggregation [ 13 , 14 ]. Therefore, protein LLPS has emerged as a new concept in bioinspired material engineering.…”

“…Lately, however, it has been demonstrated that overexpression in E. coli of certain protein sequences derived from biological materials leads to their intracellular LLPS and formation of condensates that maintain the overexpressed protein in a liquid-like state [ 14 , 24 , 25 ]. This finding provides a new opportunity to engineer protein sequences to achieve high expression yield during recombinant production while at the same time preventing their premature aggregation.…”

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

“…Although the morphologies of the structures formed by eGFP-NT2RepCT and KSI-eGFP were clearly distinct at the final time point, they could not be associated with an insoluble or condensed state exclusively based on these features. For instance, both inclusion bodies [38, 39] and liquid-like protein condensates [40, 14, 34] have been observed to locate to either one or both poles of the E. coli cell with similar morphologies. As such, we probed their material state using fluorescence recovery after photobleaching (FRAP).…”

“…LLPS provides a way to regulate proteins' transition from a soluble (liquid-like state) into the solid final form of a material. During this process the condensed phase can also serve to prevent premature aggregation [13,14].Therefore, protein LLPS has emerged as a new concept in bioinspired material engineering. In this approach proteins derived from sequences identified in biological materials are recombinantly produced, purified and assembled through LLPS into bioinspired materials in a biomimetic process.…”