Virus-like particles (VLPs) have been employed for a number of nanometric applications because they self-assemble, exhibit a high degree of symmetry, and can be genetically and chemically modified. However, high symmetry does not allow for a single unique modification site on the VLP. Here, we demonstrate the co-expression of the cytotoxic A2 protein and the coat protein of the bacteriophage Q beta to form a nearly monodispersed population of novel VLPs. Cell-free protein synthesis allows for direct access and optimization of protein-synthesis and VLP-assembly. The A2 is shown to be incorporated at high efficiency, approaching a theoretical maximum of one A2 per VLP. This work demonstrates de novo production of a novel VLP, which contains a unique site that has the potential for future nanometric engineering applications. (c) 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2012