Packaged molecular machines are available in large amounts using dual expression vectors that guide the preparation of Qβ virus-like particles encapsulating multiple copies of functional enzymes. Packaging is promoted by RNA aptamer sequences that bridge between the coat protein and a peptide tag fused to the desired cargo. Peptidase E and luciferase were thus encapsulated and shown to be catalytically active inside the particle. The encapsulated enzymes are less sensitive to inactivation by heating and surface adsorption than the corresponding free enzymes. This system represents a modular way to marry catalytic activity with robust scaffolding for the development of multifunctional materials. Keywordsvirus-like particles; Qβ bacteriophage; enzyme catalysis; enzyme protection; RNA-protein interactions; encapsulationThe sequestration of functional units from the environment is a hallmark of biological organization. In addition to encapsulation within lipid membrane-bound organelles, proteinaceous cages serve this purpose for many prokaryotes.[1] From a chemical perspective, the outstanding advantages of such packages are their capabilities for high selectivity and activity, both achieved by encapsulating only those catalysts required for the desired task in confined space, and the potential for the container to control its position in a complex environment. Artificial encapsulation or immobilization on solid supports has been shown to confer stability as well as facilitate purification and reuse.[2] While chemists have sequestered enzymes in or on a wide variety of non-biological compartments, Nature remains the undisputed master of the art.Protein nanoparticles represent a uniquely useful bridge between chemistry, materials science, and biology because they combine robust self-assembly properties with geneticallyenabled atomic control of chemical reactivity. The synthetic biomimetic packaging of functional proteins has been accomplished with several different types of protein nanoparticles. Two general strategies have been employed: genetic fusion of the cargo to a component that directs localization to the particle interior,[3] and non-specific packaging by in vitro assembly.[4] However, yields of the encapsulated protein products have been low, and, while examples of increased stability towards a variety of treatments have been noted, [3b, 3e, 4b] no quantitative kinetic comparisons of enzymes in free vs. protein-encapsulated forms have been described. We report here the use of a virus-like particle for this purpose, providing a general and robust method for the encapsulation of highly active enzymes. [8] The infectious phage particle packages its singlestranded RNA genome by virtue of a high-affinity interaction between a hairpin structure and interior-facing residues of the CP.[9] This interaction is preserved when the CP is expressed recombinantly to form VLPs[10] and we used this to direct the packaging of cargo materials (Figure 1). A related approach has been reported by Franzen and coworkers t...

The single-coat protein (CP) of bacteriophage Qβ self-assembles into T = 3 icosahedral virus-like particles (VLPs), of interest for a wide range of applications. These VLPs are very stable, but identification of the specific molecular determinants of this stability is lacking. To investigate these determinants along with manipulations that confer more capabilities to our VLP material, we manipulated the CP primary structure to test the importance of various putative stabilizing interactions. Optimization of a procedure to incorporate fused CP subunits allowed for good control over the average number of covalent dimers in each VLP. We confirmed that the disulfide linkages are the most important stabilizing elements for the capsid and that acidic conditions significantly enhance the resistance of VLPs to thermal degradation. Interdimer interactions were found to be less important for VLP assembly than intradimer interactions. Finally, a single point mutation in the CP resulted in a population of smaller VLPs in three distinct structural forms.

Qβ virus-like particles encapsulating multiple copies of fluorescent proteins were generated in high yields using a modular system enhanced by specific engineered RNA-protein interactions. The resulting particles were structurally indistinguishable from recombinant Qβ alone. The encapsidated proteins were nearly identical in photochemical properties to monomeric analogues, were more stable toward thermal degradation, and were protected from proteolytic cleavage. Residues on the outer capsid surface were chemically derivatized by acylation and azide-alkyne cycloaddition without affecting the fluorescence properties of the packaged proteins. A high affinity carbohydrate-based ligand of the CD22 receptor was thereby attached, and specific cell labeling by the particles was successfully detected by flow cytometry and confocal laser microscopy.

Bacteriophage Qβ coat protein forms uniform virus-like particles when expressed recombinantly in a variety of organisms. We have inserted the IgG-binding Z domain at the carboxy terminus of the coat protein and coexpressed this chimeric subunit with native coat protein to create hybrid, IgG-binding virus-like particles. Extracellular osmolytes were found to have an effect on the incorporation efficiency of fusion proteins into VLPs in E. coli when a carbenicillin, but not a kanamycin, selection marker was used. The addition of sucrose to the growth media decreased incorporation efficiency; the osmoprotectant glycine betaine eliminated this effect. The decrease in efficiency was not observed when carbenicillin was omitted from the final expression culture. The addition of sodium chloride instead of sucrose gave rise to particles with a larger number of fusion proteins than the standard conditions. These results illustrate that cellular conditions should be taken into account even in apparently simple systems when natural or engineered protein nanoparticles are made.

Grain yield and semolina quality traits are essential selection criteria in durum wheat breeding. However, high phenotypic screening costs limit selection to relatively few breeding lines in late generations. This selection paradigm confers relatively low selection efficiency due to the advancement of undesirable lines into expensive yield trials for grain yield and quality trait testing. Marker-aided selection can enhance selection efficiency, especially for traits that are difficult or costly to phenotype. The aim of this study was to identify major quality trait quantitative trait loci (QTL) for marker-assisted selection (MAS) and to explore potential application of genomic selection (GS) in a durum wheat breeding program. In this study, genome-wide association mapping was conducted for five quality traits using 1184 lines from the North Dakota State University (NDSU) durum wheat breeding program. Several QTL associated with test weight, semolina color, and gluten strength were identified. Genomic selection models were developed and forward prediction accuracies of 0.27 to 0.66 were obtained for the five quality traits. Our results show the potential for grain and semolina quality traits to be selected more efficiently through MAS and GS with further refinement. Considerable opportunity exists to extend these techniques to other traits such as grain yield and agronomic characteristics, further improving breeding efficiency in durum cultivar development. Durum wheat (Triticum turgidum var. durum, 2n = 4x = 28, AABB), one of the first domesticated crops, is a staple food crop with an annual global production of