Ultrasound and hyperpolarized magnetic resonance imaging enable the visualization of biological processes in deep tissues. However, few molecular contrast agents are available to connect these modalities to specific aspects of biological function. We recently discovered that a unique class of gas-filled protein nanostructures known as gas vesicles could serve as nanoscale molecular reporters for these modalities. However, the need to produce these nanostructures via expression in specialized cultures of cyanobacteria or haloarchaea limits their broader adoption by other laboratories and hinders genetic engineering of their properties. Here, we describe recombinant expression and purification of Bacillus megaterium gas vesicles using a common laboratory strain of Escherichia coli, and characterize the physical, acoustic, and magnetic resonance properties of these nanostructures. Recombinantly expressed gas vesicles produce ultrasound and hyperpolarized Xe-129 MRI contrast at subnanomolar concentrations, thus validating a simple platform for their production and engineering. (C) 2018 American Institute of Chemical Engineers