Targeting at the Nanoscale: A Novel S-Layer Fusion Protein Enabling Controlled Immobilization of Biotinylated Molecules.

Melinda Varga

doi:10.3390/nano6110199

Abstract

With the aim of constructing an S-layer fusion protein that combines both excellent self-assembly and specific ligand i.e., biotin binding ability, streptavidin (aa 16-133) was fused to the S-layer protein of Sporosarcina ureae ATCC 13881 (SslA) devoid of its N-terminal 341 and C-terminal 172 amino acids. The genetically engineered chimeric protein could be successfully produced in E. coli, isolated, and purified via Ni affinity chromatography. In vitro recrystallisation experiments performed with the purified chimeric protein in solution and on a silicon wafer have demonstrated that fusion of the streptavidin domain does not interfere with the self-assembling properties of the S-layer part. The chimeric protein self-assembled into multilayers. More importantly, the streptavidin domain retained its full biotin-binding ability, a fact evidenced by experiments in which biotinylated quantum dots were coupled to the fusion protein monomers and adsorbed onto the in vitro recrystallised fusion protein template. In this way, this S-layer fusion protein can serve as a functional template for the controlled immobilization of biotinylated and biologically active molecules.

Highlights

Biomolecular templating is fundamental in the development of advanced biosensors, bioreactors, affinity chromatographic separation materials, and many diagnostics such as those used in cancer therapeutics [1,2,3]
The DNA encoding the SslA341-925streptavidin fusion protein was created by overlap extension polymerase chain reaction (PCR)
A previous study about this S-layer protein showed that the central part of SslA is sufficient for in vitro self-assembly into monolayers exhibiting the p4 lattice structure [17]

Summary

Introduction

Biomolecular templating is fundamental in the development of advanced biosensors, bioreactors, affinity chromatographic separation materials, and many diagnostics such as those used in cancer therapeutics [1,2,3]. In this regard, biomolecules are used to precisely position nanoscale materials onto substrates. Bacterial cell surface layers (S-layers) are two dimensional protein lattices that cover the cell surface of many Bacteria and Archaea [4]. These lattices are composed of identical (glyco)protein subunits, exhibit oblique (p1, p2), square (p4), or hexagonal (p3, p6) symmetry and are very porous. Due to the monomolecular structure of the lattices, the pores are identical in size and morphology, ranging between 2 and 8 nm [5]

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Conclusion