Genetically programmed in vivo packaging of protein cargo and its controlled release from bacteriophage P22

Viruses have evolved exquisite selectivity for the complex hierarchical assembly of components directed towards the programmed packaging and sequestration of cargo molecules. Furthermore, protein-based containers are increasingly recognized for their ability to act as isolated intracellular compartments with unique and selective catalytic functionality. The inspiration drawn from these systems has provided blueprints for synthetic encapsulation approaches based on programmed interactions in self-assembling nanocontainer systems. Heterologously expressed protein-cage architectures are excellent candidates for such engineered systems, owing to their self-assembly from a limited number of protein subunits into symmetrical, highly monodisperse structures, the ability to package or release the cargo, and the relative ease of large-scale production and purification. This approach has been used with icosahedral viral capsids to encapsulate enzymes, other proteins, and metal cores. Furthermore, a wide range of other protein-cage architectures have been used to incorporate active sites and/or fluorescent proteins and to develop in vivo selection for packaging based on the cellular toxicity of free (unpackaged) proteases. The design of effective, widely applicable nanocontainers for programmed cargo encapsulation requires a capsid structure with sufficient internal volume for the cargo of interest to be loaded in high copy number, physical stability over a wide range of conditions, and the incorporation of molecular recognition for both specific packaging of the cargo and programmed assembly. A number of approaches have been developed for programmed encapsulation; however, in all cases the copy number of the encapsulated cargo has been relatively modest. Herein we report an approach for the programmed encapsulation of gene-product fusions in very high copy number through the use of the capsid and scaffoldprotein machinery from the bacteriophage P22 in a heterologous expression system. We used the encapsulation of a fluorescent protein as a proof of concept. Having established the methodology for genetically programmed self-assembly and encapsulation, we will be able to use the well-designed multiple cloning site described below to readily substitute a wide range of gene products into a fusion site for programmed encapsulation. Our genetically engineered system is based on the Salmonella typhimuriam bacteriophage P22, which assembles from 420 copies of the coat-protein subunit into an icosahedral capsid with the assistance of 100–330 copies of a scaffold protein (SP). The C terminus of the SP interacts with the coat protein and is necessary for self-assembly, whereas the N terminus can be severely truncated or mutated with little to no effect on assembly. Our system genetically fuses cargo proteins to a truncated form of the P22 scaffold protein, which acts as a template for capsid assembly as well as a specific encapsulation signal for the cargo. In this way, additional space within the capsid from the truncation of the SP enables the cargo to be assembled in vivo with high packaging efficiency. This method does not alter the coat protein in any way. It eliminates the need for affinity tags, chemical linkers, or bridging ligands, and perhaps avoids the misassembly of virus particles. Instead, the fusion of the scaffold protein to the cargo naturally provides the necessary affinity to the interior of the capsid, and assembly is templated around the cargo. Through genetic manipulation of the P22 assembler plasmid, a vector containing the gene for P22 coat protein and a truncated variant of the wild-type (WT) scaffold protein encoding for amino acids 141–303 (SP141) was designed. A thrombin-cleavage site and a newmultiple cloning site (MCS) were included upstream of the SP141 gene. (Figure 1a; see also Figure S1 in the Supporting Information). This new MCS enabled the insertion of a series of fluorescent-protein genes (enhanced green fluorescent protein (EGFP) and mCherry) for the creation of N-terminal fluorescentprotein–SP141 fusions. In these protein fusions, the thrombin-cleavage site is located between SP141 and the fluorescent protein and acts as both a flexible linker sequence and an accessible cleavage site for the release of the fluorescent protein from the SP141 as needed. As expected, the P22 fluorescent-protein–SP141 procapsids were indistinguishable from the WT P22 procapsids during the expression and purification process except for their brilliant color, which indicated the expression, encapsulation, and proper folding of the fluorescent protein (Figure 1b). On average, the expression yields were 150 mg of P22 with encapsulated fluorescent cargo per liter of Escherichia coli culture after a single purification step by sucrose-cushion ultracentrifugation. Assembly of the capsids from the SP141 [*] A. O’Neil, C. Reichhardt, B. Johnson, Dr. T. Douglas Chemistry and Biochemistry Department Center for Bio-Inspired Nanomaterials, Montana State University Bozeman, MT 59717 (USA) Fax: (+1)406-994-5407 E-mail: tdouglas@chemistry.montana.edu