Novel Biocatalysts Based on S‐Layer Self‐Assembly of Geobacillus Stearothermophilus NRS 2004/3a: A Nanobiotechnological Approach

Abstract

The crystalline cell-surface (S) layer sgsE of Geobacillus stearothermophilus NRS 2004/3a represents a natural protein self-assembly system with nanometer-scale periodicity that is evaluated as a combined carrier/patterning element for the conception of novel types of biocatalyst aiming at the controllable display of biocatalytic epitopes, storage stability, and reuse. The glucose-1-phosphate thymidylyltransferase RmlA is used as a model enzyme and chimeric proteins are constructed by translational fusion of rmlA to the C-terminus of truncated forms of sgsE (rSgsE , rSgsE ) and used for the construction of three principal types of biocatalysts: soluble (monomeric), self-assembled in aqueous solution, and recrystallized on negatively charged liposomes. Enzyme activity of the biocatalysts reaches up to 100% compared to sole RmlA cloned from the same bacterium. The S-layer portion of the biocatalysts confers significantly improved shelf life to the fused enzyme without loss of activity over more than three months, and also enables biocatalyst recycling. These nanopatterned composites may open up new functional concepts for biocatalytic applications in nanobiotechnology.

Recombinant Glycans on an S‐Layer Self‐Assembly Protein: A New Dimension for Nanopatterned Biomaterials

“…The length of the base vectors, however, compared well with previous results of negatively stained native S-layer glycoprotein from G. stearothermophilus NRS 2004/3a [18] as well as with the lattice parameters of selfassembled rSgsE protein (a = 11.6 nm, b = 9.4 nm, γ ≈ 78°). [2] Comparing that material with the S-layer neoglycoprotein produced in the course of the present study, a slightly changed staining behavior of the latter was observed on the micrographs, with the lattice of the neoglycoprotein apparently blurred to some extent. Despite this fact, in self-assembled A_SgsE_T12-pgl the SgsE S-layer protein matrix itself kept its crystallinity.…”

“…Either form of SgsE has been shown to possess an excellent self-assembly capability. [2] G_SgsE was included in this study because it can be imagined that, due to its smaller size compared to the mature protein, its export to the periplasm will be facilitated. In another neoglycoprotein production experiment, ssPelB and ssTorA were successfully used for the transfer of the N-glycosylation target protein AcrA of C. jejuni to the periplasm of E. coli, demonstrating that bacterial N-glycosylation can occur independently of the protein translocation machinery.…”

“…Self-Assembly of SgsE Neoglycoprotein Carrying the C. jejuni Heptasaccharide After the glycoprotein nature of the A_SgsE_T12-pgl neoglycoprotein was confirmed, selfassembly of the recombinant construct was investigated. Following a protocol elaborated in the course of a recent study, [2] self-assembly of the monomeric S-layer neoglycoprotein as obtained after purification was triggered upon removal of the chaotropic agent, by dialysis against distilled water. Self-assembly was investigated by electron microscopy using negatively stained preparations.…”

“…Crystalline-cell surface layers (S-layers) of prokaryotic organisms are a very potent self-assembly system, which can be used in bottom-up processes as a patterning element for a "biomolecular construction kit". [1][2][3][4][5] The unique property of isolated S-layer protein subunits (monomers) to reassemble into two-dimensional (2D) crystals that are identical to those found on intact bacterial cells, either in suspension, on diverse solid supports, on liposomes, or on various interfaces, opens a wide range of S-layer-based applications. [6][7][8][9] In previous studies, it has been demonstrated that several functional domains introduced into S-layer proteins at distinct positions by genetic engineering do not interfere with the intrinsic self-assembly property of the S-layer system, while simultaneously fully retaining their specific biological properties.…”

“…[6][7][8][9] In previous studies, it has been demonstrated that several functional domains introduced into S-layer proteins at distinct positions by genetic engineering do not interfere with the intrinsic self-assembly property of the S-layer system, while simultaneously fully retaining their specific biological properties. [2,4,10] A novel line of development is directed toward the integration of carbohydrate compounds (glycans) into this biomolecular construction kit through co-or post-translational fusion of the carbohydrates to the self-assembly S-layer protein matrix, leading to the production of self-assembled S-layer neoglycoproteins. [11] Considering that glycans are ubiquitous biomolecules, which, in many cases, are key to protein function, [12][13][14] it is evident that glycans are useful means for addressing various questions in the fields of basic and applied research, relating to the areas of biomimetics, drug targeting, vaccine design, [15] or diagnostics.…”