In vivo self-assembly of fluorescent protein microparticles displaying specific binding domains.

In this study, fluorescent proteins (FPs) were engineered to self-assemble into protein particles inside recombinant Escherichia coli while mediating the display of various protein functionalities such as maltose binding protein or IgG binding domains of Protein A or G, respectively. Escherichia coli produced functional FP particles of up to 30% of cellular dry weight. The use of respective FP particles displaying certain binding domains in diagnostics and as bioseparation resins was demonstrated by direct comparison to commercial offerings. It was demonstrated that variable extensions (AVTS, FHKP, LAVG, or TS) of the N-terminus of FPs (GFP, YFP, CFP, HcRed) in combination with large C-terminal extensions such as translational fusion of the polyester synthase from Ralstonia eutropha or an aldolase from Escherichia coli led to extensive intracellular self-assembly of strongly fluorescent fusion protein particles of oval shape (0.5×1 µm). The strong fluorescent label of these bioparticles in combination with covalent display of protein functions provides a molecular toolbox for the design of self-assembled microparticles suitable for antibody-capture or ligand binding based diagnostic assays as well as the high affinity purification of target compounds such as antibodies.

Ff-nano, short functionalized nanorods derived from Ff (f1, fd, or M13) filamentous bacteriophage.

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.