S‐Layers as a basic building block in a molecular construction kit

Abstract

Crystalline arrays of protein or glycoprotein subunits forming surface layers (S-layers) are the most common outermost envelope components of prokaryotic organisms (archaea and bacteria). The wealth of information on the structure, chemistry, genetics, morphogenesis, and function of S-layers has revealed a broad application potential. As S-layers are periodic structures, they exhibit identical physicochemical properties for each molecular unit down to the subnanometer level and possess pores of identical size and morphology. Many applications of S-layers in nanobiotechnology depend on the ability of isolated subunits to recrystallize into monomolecular lattices in suspension or on suitable surfaces and interfaces. S-Layer lattices can be exploited as scaffolding and patterning elements for generating more complex supramolecular assemblies and structures, as required for life and nonlife science applications.

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

“…For the first time, an S-layer neoglycoprotein has been designed, comprising the S-layer protein SgsE from G. stearothermophilus NRS 2004/3a, including the PelB signal peptide for periplasmic targeting, and the heptasaccharide Glc(GalNAc) 5 Bac of C. jejuni. For this purpose, the plasmid pACYCpgl harboring the complete pgl gene cluster of C. jejuni that is responsible for heptasaccharide biosynthesis and its transfer to the protein was transformed into E. coli BL21 Star (DE3).…”

“…In C. jejuni, Glc(GalNAc) 5 Bac is naturally linked to distinct N-glycosylation sites of the protein AcrA, [21] while the S-layer protein SgsE possesses O-glycosidically linked glycan chains. Inspection of the S-layer protein sequence revealed the presence of one putative Nglycosylation site at position N 879 (DVNQT; the glycosylated asparagine residue is underlined), conforming with the amino acid sequence requirement of the oligosaccharyl:protein transferase PglB of C. jejuni, which is the key enzyme for protein Nglycosylation.…”

“…Inspection of the S-layer protein sequence revealed the presence of one putative Nglycosylation site at position N 879 (DVNQT; the glycosylated asparagine residue is underlined), conforming with the amino acid sequence requirement of the oligosaccharyl:protein transferase PglB of C. jejuni, which is the key enzyme for protein Nglycosylation. [22,28,35] Thus, in a first glycosylation approach, the potential of PglB to glycosylate native SgsE with its endogenous heptasaccharide Glc(GalNAc) 5 Bac substrate in E. coli was investigated by expression of ssPelB-A_SgsE from plasmid pET22b-A_SgsE in E. coli BL21 Star (DE3) harboring pACYCpgl. [21] However, no glycosylated protein was visible on a Western immunoblot, implying that the putative N-glycosylation site of SgsE at position N 879 was not recognized by PglB (not shown).…”

“…Mass spectrometry was performed in order to confirm that the complete Glc(GalNAc) 5 Bac N-glycan had been transferred onto the N-glycosylation site of A_SgsE_T12. Purified A_SgsE_T12-pgl neoglycoprotein was digested with trypsin and the resulting peptides were analyzed by nanoliquid-chromatography (nano-LC) matrix-assisted laser-desorption ionization-time of flight (MALDI TOF)/TOF mass spectrometry (MS).…”

“…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.…”