| Virus-like particles are useful nanoscale scaffold reagents and have found success as a new approach to vaccination in addition to opening possibilities in drug delivery and medical imaging. We have developed techniques to produce novel virus-like protein nanoparticles displaying protein domains on their surface in addition to encapsidating heterologous proteins on their interior. Both of these techniques take advantage of the ability of the bacteriophage Qbeta coat protein to self-assemble into stable capsids in vivo and do not require in vitro post-purification assembly steps. During the development of these techniques, we have made observations indicative of multiple layers of complexity in the assembly of this nanoparticle that raise questions about the state of the Escherichia coli cell during recombinant protein production. In the synthesis of hybrid nanoparticles, we observe changes in the materials produced that are dependent on extracellular osmolytes present during nanoparticle assembly, but only in the presence of carbenicillin even when beta-lactamase is still present. During the assembly of ternary nanoparticle complexes that encapsidate cargo proteins via RNA-mediated interactions we observe remarkable variation in encapsidation copy number that can only be stabilized by defined minimal media. We have only just begun to isolate the sources of this variation, and there appears to be a complex interrelationship between the protein being encapsidated, the programmed biopolymer interactions, and the bulk E. coli cellular contents. Together, these observations suggest a framework for a more informed approach to developing E. coli as a platform for the synthesis and assembly of protein nanoparticles. |
