Abstract
F-specific filamentous phage of Escherichia coli (Ff: f1, M13, or fd) are long thin filaments (860 nm × 6 nm). They have been a major workhorse in display technologies and bionanotechnology; however, some applications are limited by the high length-to-diameter ratio of Ff. Furthermore, use of functionalized Ff outside of laboratory containment is in part hampered by the fact that they are genetically modified viruses. We have now developed a system for production and purification of very short functionalized Ff-phage-derived nanorods, named Ff-nano, that are only 50 nm in length. In contrast to standard Ff-derived vectors that replicate in E. coli and contain antibiotic-resistance genes, Ff-nano are protein-DNA complexes that cannot replicate on their own and do not contain any coding sequences. These nanorods show an increased resistance to heating at 70∘C in 1% SDS in comparison to the full-length Ff phage of the same coat composition. We demonstrate that functionalized Ff-nano particles are suitable for application as detection particles in sensitive and quantitative “dipstick” lateral flow diagnostic assay for human plasma fibronectin.
Highlights
Filamentous bacteriophages are filament-like bacterial viruses (Day, 2011; Rakonjac et al, 2011)
The helper phage Rnano3, in contrast to the original helper phage R474 used for short phage production (Specthrie et al, 1992), is a helper and a phage display vector
Rnano3 was designed for display of proteins as fusions with phage protein pIII that is present in up to five copies at one end of the Ff-derived particles
Summary
Filamentous bacteriophages are filament-like bacterial viruses (Day, 2011; Rakonjac et al, 2011). The major coat protein pVIII, that forms the shaft of the filament, is present in the virion in a few thousand copies. Two pairs of minor coat proteins, pIII/pVI and pVII/pIX, form two asymmetric ends of the filament. They are each present in the virion in up to five copies. Infection with filamentous phage is mediated by binding of a minor protein, pIII, to the primary and secondary receptors, the tip of the F-pilus and the TolQRA protein complex spanning the periplasm and the inner membrane, respectively. TolQRA, a conserved protein complex in Gram-negative bacteria, appears to be both an essential and universally required protein for filamentous phage infection, allowing what appears to be a low-efficiency broad-spectrum infection of Gram-negative bacteria by this group of phage (Heilpern and Waldor, 2000)
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