We have developed methods for producing viral-based protein cages in high yield that are amenable to genetic modification. Expression of the structural protein of Cowpea chlorotic mottle bromovirus (CCMV) using the yeast-based Pichia pastoris heterologous expression system resulted in the assembly of particles that were visibly indistinguishable from virus particles produced in the natural host. We have shown that a collection of non-infectious CCMV coat protein mutants expressed in the P. pastoris system assemble into viral protein cages with altered architectures and function. This provides an alternative to other heterologous expression systems for production of viral structural proteins in which expression has resulted in unassembled cages. Heterologous expression in P. pastoris further enhances the development of viral-based protein cages as biotemplates for nanotechnology and for future studies examining details of icosahedral virus assembly.