Virus-like particles (VLPs) have been extensively explored as nanoparticle vehicles for many applications in biotechnology (e.g., vaccines, drug delivery, imaging agents, biocatalysts). However, amino acid sequence plasticity relative to subunit expression and nanoparticle assembly has not been explored. Whereas the hepatitis B core protein (HBc) VLP appears to be the most promising model for fundamental and applied studies; particle instability, antigen fusion limitations, and intrinsic immunogenicity have limited its development. Here, we apply Escherichia coli-based cell-free protein synthesis (CFPS) to rapidly produce and screen HBc protein variants that still self-assemble into VLPs. To improve nanoparticle stability, artificial covalent disulfide bridges were introduced throughout the VLP. Negative charges on the HBc VLP surface were then reduced to improve surface conjugation. However, removal of surface negative charges caused low subunit solubility and poor VLP assembly. Solubility and assembly as well as surface conjugation were greatly improved by transplanting a rare spike region onto the common shell structure. The newly stabilized and extensively modified HBc VLP had almost no immunogenicity in mice, demonstrating great promise for medical applications. This study introduces a general paradigm for functional improvement of complex protein assemblies such as VLPs. This is the first study, to our knowledge, to systematically explore the sequence plasticity of viral capsids as an approach to defining structure function relationships for viral capsid proteins. Our observations on the unexpected importance of the HBc spike tip charged state may also suggest new mechanistic routes toward viral therapeutics that block capsid assembly.virus-like particle | engineered nanoparticles | disulfide stabilization | hepatitis core protein | cell-free protein synthesis V irus-like particles (VLPs) are probably the most precisely defined and, therefore, potentially the most useful complex nanometer-scale scaffolds (1). VLPs mimic the capsid structure of real viruses, but lack infectious genetic material. Selected VLPs derived from pathogens have already provided major advances in the development of vaccines that have known and relatively homogeneous structures as well as enhanced immunogenicity (2). Such nanoparticles provide comparable cellular uptake and intracellular trafficking compared with natural viruses (3), and also have repetitive surfaces for the high-density display of vaccine antigens (4). In addition, VLPs offer favorable trafficking from the injection site to lymph nodes (5). Since the first reported use of a hepatitis B core protein (HBc) VLP as an antigen carrier in 1987 (6), at least 110 VLP vaccine candidates have been constructed by using capsid proteins from 35 different viral families (7).Among different types of VLPs, the HBc VLP is the most flexible and promising model for fundamental and applied immunological studies (8). One advantage of the HBc VLP platform is that the capsids can be produ...