Plant-made virus-like particles (VLPs), composed of HIV-1 Gag and deconstructed gp41 proteins, have been shown to be safe and immunogenic in mice. Here, we report the successful production of HIV-1 Gag/dgp41 VLPs in Nicotiana benthamiana, using an enhanced geminivirus-based expression vector. This novel vector results in unique expression kinetics, with peak protein accumulation and minimal necrosis achieved on day 4 post-infiltration. In comparing various purification strategies, it was determined that a 20% ammonium sulfate precipitation is an effective and efficient method for removing plant proteins and purifying the recombinant VLPs of interest. If further purification is required, this may be achieved through ultracentrifugation. VLPs are a useful platform for a variety of biomedical applications and developing the technology to efficiently produce VLPs in the plant expression system is of critical importance.