Small surfactant-like peptides can drive soluble proteins into active aggregates

Abstract

Background: Inactive protein inclusion bodies occur commonly in Escherichia coli (E. coli) cells expressing heterologous proteins. Previously several independent groups have found that active protein aggregates or pseudo inclusion bodies can be induced by a fusion partner such as a cellulose binding domain from Clostridium cellulovorans (CBDclos) when expressed in E. coli. More recently we further showed that a short amphipathic helical octadecapeptide 18A (EWLKAFYEKVLEKLKELF) and a short beta structure peptide ELK16 (LELELKLKLELELKLK) have a similar property.

Results: In this work, we explored a third type of peptides, surfactant-like peptides, for performing such a "pulling-down" function. One or more of three such peptides (L6KD, L6K2, DKL6) were fused to the carboxyl termini of model proteins including Aspergillus fumigatus amadoriase II (AMA, all three peptides were used), Bacillus subtilis lipase A (LipA, only L6KD was used, hereinafter the same), Bacillus pumilus xylosidase (XynB), and green fluorescent protein (GFP), and expressed in E. coli. All fusions were found to predominantly accumulate in the insoluble fractions, with specific activities ranging from 25% to 92% of the native counterparts. Transmission electron microscopic (TEM) and confocal fluorescence microscopic analyses confirmed the formation of protein aggregates in the cell. Furthermore, binding assays with amyloid-specific dyes (thioflavin T and Cong red) to the AMA-L6KD aggregate and the TEM analysis of the aggregate following digestion with protease K suggested that the AMA-L6KD aggregate may contain structures reminiscent of amyloids, including a fibril-like structure core.

Conclusions: This study shows that the surfactant-like peptides L6KD and it derivatives can act as a pull-down handler for converting soluble proteins into active aggregates, much like 18A and ELK16. These peptide-mediated protein aggregations might have important implications for protein aggregation in vivo, and can be explored for production of functional biopolymers with detergent or other interfacial activities.

Figure 1

Schematics for surfactant-like peptide and fusion protein constructs. (A) The sequences of the surfactant-like peptides used in this study. (B) Genetic constructs of the surfactant-like peptide fusion proteins. Four model proteins: Aspergillus fumigatus amadoriase II (AMA), Bacillus subtilis lipase A (LipA), Bacillus pumilus xylosidase (XynB), and green fluorescent protein (GFP); linker, PTPPTTPTPPTTPTPTP.

Figure 2

Distributions of enzyme activities in the soluble and insoluble fractions of cell lysates. (B) AMA-native and AMA-L₆KD. (B) LipA-native and LipA-L₆KD. (C) XynB-native and XynB-L₆KD. The activities were determined using three independent experiments and normalized to the total activities of the respective native enzyme extracted from a same amount of cells (OD₆₀₀). Standard deviations are also shown.

Figure 3

Intracellular localization of fusion proteins in E. coli. (A) and (B), TEM microscopic images for AMA-native and AMA-L₆KD, respectively. (C) Confocal fluorescent micrograph for GFP-L₆KD. Size bars are also shown.

Figure 4

Binding of AMA-L₆KD aggregate to thioflavin T (ThT). The histograms show the fluorescence intensity of ThT at 480 nm (excited at 445 nm) in the presence and in the absence of AMA-L₆KD aggregate, in arbitrary units (a. u.).

Figure 5

Fibrillar structure of AMA-L₆KD protein aggregate. The micrograph shows the fibers of AMA-L₆KD aggregate after proteolytic treatment by protease K. Size bar is also shown.