Characterization of a Bacterial Self-Assembly Surface Layer Protein and Its Application as an Electrical Nanobiosensor

Abstract

Bacterial cell surface layers (S-layers) are one of the most common outermost cell envelope components of prokaryotic organisms (Archaea and bacteria), which can self-assemble into two-dimensional (2D) crystalline arrays. Production and characterization of the bacterial S-layer protein (SLP) from Geobacillus stearothermophilus, a thermophilic bacterium, are demonstrated in this study. Based on this, purified SLPs were applied for wrapping around single-walled carbon nanotubes (CNTs) and applying as electrochemical sensing tools. For the large scale production of SLP, fed-batch culture of G. stearothermophilus was carried out by DO-stat strategy. Purified SLPs were characterized by atomic force microscopy (AFM). After recrystallization of purified SLPs with gold colloids, the formation of two-dimensional (2D) oblique lattice was observed by transmission electron microscopy (TEM). Metallic or near metallic characteristics of CNTs were measured by current-voltage (I-V) analyzer. By high-cell density cultivation, cells grew to 10 g/l of dry cell weight in 65 h and the S-layer contents were achieved up to 40% of total proteins. The SLPs were purified to electrophoretic homogeneity and the molecular mass was estimated to be about 105 kDa. The purified SLPs were self-assembled and confirmed their hexagonal symmetry lattice structure. The SLP subunits were cross-linked to each other and to the underlying CNTs by non-covalent interaction, which was caused to change the electric current between natural CNTs and SLP-wrapped CNTs. One-dimensional structure and large aspect ratio of the functionalized CNTs may allow effective targeting of biomolecules by specific binding, such as protein-protein, DNA-DNA, and protein-ligand interaction. Bacterial SLP could be used as a biological template for immobilization molecular array, and provides new approaches for nanoelectronic biosensor applications.