Abstract
Tobacco mosaic virus (TMV) is a robust nanotubular nucleoprotein scaffold increasingly employed for the high density presentation of functional molecules such as peptides, fluorescent dyes, and antibodies. We report on its use as advantageous carrier for sensor enzymes. A TMV mutant with a cysteine residue exposed on every coat protein (CP) subunit (TMVCys) enabled the coupling of bifunctional maleimide-polyethylene glycol (PEG)-biotin linkers (TMVCys/Bio). Its surface was equipped with two streptavidin [SA]-conjugated enzymes: glucose oxidase ([SA]-GOx) and horseradish peroxidase ([SA]-HRP). At least 50% of the CPs were decorated with a linker molecule, and all thereof with active enzymes. Upon use as adapter scaffolds in conventional “high-binding” microtiter plates, TMV sticks allowed the immobilization of up to 45-fold higher catalytic activities than control samples with the same input of enzymes. Moreover, they increased storage stability and reusability in relation to enzymes applied directly to microtiter plate wells. The functionalized TMV adsorbed to solid supports showed a homogeneous distribution of the conjugated enzymes and structural integrity of the nanorods upon transmission electron and atomic force microscopy. The high surface-increase and steric accessibility of the viral scaffolds in combination with the biochemical environment provided by the plant viral coat may explain the beneficial effects. TMV can, thus, serve as a favorable multivalent nanoscale platform for the ordered presentation of bioactive proteins.
Highlights
Biological scaffolds have been used for the spatially precise immobilization and presentation of organic or inorganic materials and functional molecules increasingly during the past decades, due to their regular shapes, multivalence on the nanometer scale and self-assembly capabilities
Catalytic activities of adapter-bound enzyme conjugates (i.e., [SA]-glucose oxidase (GOx) and [SA]-horseradish peroxidase (HRP) displayed on TMVCys/Bio sticks, CPCys/Bio aggregates, or biotin linker molecules, respectively) adsorbed to solid microtiter plate supports were compared to those of the same enzyme conjugates bound to the plates without any adapters (“free”)
A 22-fold molar excess of biotin linker relative to the viral CPCys subunits modified approximately 50% of the target sites, as determined by densitometry, and this ratio was applied further on
Summary
Biological scaffolds have been used for the spatially precise immobilization and presentation of organic or inorganic materials and functional molecules increasingly during the past decades, due to their regular shapes, multivalence on the nanometer scale and self-assembly capabilities. Several virions are extremely stable, can be produced in large quantities and modified genetically as well as chemically (for recent reviews, see Soto and Ratna, 2010; Liu et al, 2012b; Bittner et al, 2013; Bernard and Francis, 2014; Culver et al, 2015) Since they are highly promising building blocks to construct novel nanostructured materials, plant viruses have served as scaffolds for a wide range of functional molecules such as reporter dyes (e.g., Cruz et al, 1996; Gillitzer et al, 2002; Lewis et al, 2006; Martin et al, 2006), antigens for vaccination purposes as reviewed in detail (e.g., Chackerian, 2007; Crisci et al, 2012; Kushnir et al, 2012), antibodies as tracers in immunoassays (Sapsford et al, 2006) or immunoadsorbents (Werner et al, 2006), medical imaging reagents or drugs (with numerous examples described in Yildiz et al, 2011; Khudyakov and Pumpens, 2015), and a plentitude of inorganic and synthetic compounds to fabricate technically applicable hybrid materials and devices with novel physical and chemical properties (reviewed in Lee et al, 2012b; Bittner et al, 2013; Li and Wang, 2014; Love et al, 2014; Culver et al, 2015). An emerging field is the immobilization of proteins conferring complex functionalities, including e.g., receptor- or hapten-binding modules and enzymes (e.g., Chatterji et al, 2004; Carette et al, 2007; Soto et al, 2009; Szuchmacher Blum et al, 2011; Aljabali et al, 2012; Cardinale et al, 2012, and references therein; Pille et al, 2013)
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