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.

Keywords: E. coli; amyloids; liquid-liquid phase separation; membraneless organelles; protein condensates; protein tag; recombinant expression.

FIGURE 1

Schematic illustration of the construct, expression, and purification process of BEAK‐tagged amyloidogenic peptides. (a) BEAK‐tag—designed based on HBP‐1 sequence (upper panel, adapted from Gabryelczyk et al.). In the BEAK‐tag a part of modular region (initiating LLPS) was substituted with the target sequence; however, the fusion protein retained its ability to undergo LLPS. Protease recognition site (trypsin/TEV) was inserted between the BEAK‐tag and the target sequence. (b) Expression of the eGFP‐tagged fusion proteins leads to the formation of intracellular condensates that can be visualized with fluorescence microscopy. (c) Purification protocol based on low pH extraction from the soluble fraction. The Aβ‐P3* peptide can be obtained without additional chromatography purification (as described in the lower‐right box)

FIGURE 2

Fluorescence microscopy and phase‐contrast images of bacterial cells expressing eGFP‐BEAK‐tag‐TGFBIp1 fusion protein. (a) Fluorescent protein condensates localized near the polar regions of the cells indicate the formation of membraneless organelles. (b) Diffusion of the fluorescent signal indicating liquid‐like properties of the protein condensates

FIGURE 3

Overlay of the ¹H‐¹⁵ N‐HSQC NMR spectra of the BEAK‐tag (without fused peptide) with (a) BEAK‐tag‐TGFBIp1 (b) BEAK‐tag‐TGFBIp2 fusion proteins. Cross‐peaks assigned to the TGFBIp1 and 2 peptides marked with “x.” The chemical shifts of the target peptides in the fusion construct correspond to those observed in the synthetically produced version and exhibited uniform intensity, indicating the absence of secondary structure in the fused peptide as well as the random coil nature of the conformational ensemble

FIGURE 4

LLPS of the BEAK‐tag‐TGFBIp1 fusion protein in vitro in a molecular environment mimicking the cytoplasm of E. coli. (a) Phase‐contrast optical microscopy image of microdroplets (condensed protein phase). (b) Time‐lapse images showing fusion of microdroplets. The whole process is presented in Movie S1. (c) Adhesion and spreading of the microdroplets on a glass surface. (d) Fluorescence microscopy imaging of protein microdroplets using the DroProbe fluorescent dye that acts as a molecular probe for LLPS. Microdroplets in the contrast microscopy image (center) fully overlap with those from the fluorescent image, confirming that the microdroplets are liquid‐like formed by LLPS

FIGURE 5

TEM imaging and ssNMR of the amyloid fibrils formed by amyloidogenic peptides: (a) TGFBIp1, (b) TGFBIp2, (c) Aβ‐P3*, (d) TGFBIp2 (after TEV cleavage). (e) 2D ¹³C—¹³C chemical shift correlation DARR experiment of ¹³C, ¹⁵N‐labeled TGFBIp2 measured at 600 MHz using the 1.9 mm MAS HXY probe. Inset: ¹H—¹³C cross‐polarization (CP), MAS NMR spectra of recombinant (blue) and synthetic TGFBIp2 (red) peptides