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

Abstract

BackgroundInactive 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.ResultsIn 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.ConclusionsThis 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.

Self-assembly amphipathic peptides induce active enzyme aggregation that dramatically increases the operational stability of nitrilase

“…19,21,22 Here, the amphipathic 18A (EWLKA-FYEKVLEKLKELF) short peptide was attached to the C-terminus of a nitrilase to investigate the formation of inclusion bodies and the impact this process had on the catalytic properties of the nitrilase.…”

Sheltering DNA in self-organizing, protein-only nano-shells as artificial viruses for gene delivery

“…8 Complexes of DNA and cationic proteins often result in polydisperse soluble aggregates probably derived from intrinsically disordered protein-protein interactions, 9,10 or in which the DNA itself plays a leading architectonic role, stabilizing aggregation-prone protein monomers in the form of monodisperse nanoparticles. 11 Self-assembling peptides that organize as different types of nanostructured materials, 12 promote unspecific aggregation when fused to larger proteins, 13,14 making them useless as fine architectonic tags. In summary, the rational de novo design of protein monomers with self-assembling activities has remained so far unreachable.…”

Why and how protein aggregation has to be studied in vivo

“…This approach has been applied, for instance, to investigate the presence of functional proteins embedded in bacterial IBs [22-24]. Interestingly, in recent works it has been observed that the fusion of self-assembling or surfactant-like peptides to different proteins makes it possible to obtain active IBs, whose formation was detected in vivo monitoring the fluorescence of GFP - taken as a model system - fused to the peptide.…”

“…Indeed, the bacterial cell images, obtained by confocal microscopy, showed a diffuse fluorescence when GFP was expressed alone, in a soluble form. When, instead, the GFP was expressed fused to the self-assembling or surfactant-like peptide, the fluorescence appeared localized in the cell, indicating the formation of active IBs [23,24]. Moreover the use of GFP tag as a reporter for corrected folding has been employed for the screening of Aβ mutations and chemical compounds able to tune the aggregation propensity of the peptide.…”