Functionalized Protein Nanotubes Based on the Bacteriophage vB_KleM-RaK2 Tail Sheath Protein

Abstract

We report on the construction of functionalized nanotubes based on tail sheath protein 041 from vB_KleM-RaK2 bacteriophage. The truncated 041 protein (041Δ200) was fused with fluorescent proteins GFP and mCherry or amidohydrolase YqfB. The generated chimeric proteins were successfully synthesized in E. coli BL21 (DE3) cells and self-assembled into tubular structures. We detected the fluorescence of the structures, which was confirmed by stimulated emission depletion microscopy. When 041Δ200GFP and 041Δ200mCherry were coexpressed in E. coli BL21 (DE3) cells, the formed nanotubes generated Förster resonance energy transfer, indicating that both fluorescent proteins assemble into a single nanotube. Chimeric 041Δ200YqfB nanotubes possessed an enzymatic activity, which was confirmed by hydrolysis of N4-acetyl-2′-deoxycytidine. The enzymatic properties of 041Δ200YqfB were similar to those of a free wild-type YqfB. Hence, we conclude that 041-based chimeric nanotubes have the potential for the development of delivery vehicles and targeted imaging and are applicable as scaffolds for biocatalysts.

Nanotubes from bacteriophage tail sheath proteins: internalisation by cancer cells and macrophages

“…The retention of enzymatic activity in nanostructures formed by fused recombinant proteins was also demonstrated in our previous study with a bacteriophage tail sheath protein derived from the vB_KleM-RaK2 bacteriophage, 31 confirming that proteinaceous nanotubes are a suitable carrier for enzymes.…”

“…Our recent study has shown that the fusion of an amidohydrolase YqfB to a nanotube forming monomer 041Δ200 resulted in a 3-fold decrease in enzymatic activity. 31 Therefore, at least a partial impairment in the activity of SNAP-tag within the nanotubes can be anticipated, but there was sufficient binding of the fluorescent enzyme substrate to observe the nanotubes within the cells.…”

“…The cells were disrupted by sonication and the soluble protein fraction was purified as described previously. 31 …”

“…TEM analysis was performed as described previously. 31 The particle size was estimated using ImageJ (FIJI) software 63 choosing the sample size n = 30.…”

“…Soluble cell-free extract (25 μl) was labelled with SNAP-Surface Alexa Fluor 488, diluted 10×, and subjected to high-performance liquid chromatography (HPLC) with a UV-vis and fluorescence detection system (Shimadzu, Kyoto, Japan). A TSKgel G4000SWXL column was used for analysis as described previously, 31 equilibrated in 10 mM Tris–HCl, pH 8.0.…”