Abstract
The continuing emergence of variants of the SARS-CoV-2 virus requires the development of modular molecular therapies. Here, we engineered a recombinant amphiphilic protein, oleosin, to spontaneously self-assemble into multivalent micellar nanostructures which can block the Spike S1 protein of SARS-CoV-2 pseudoviruses (PVs). Short recombinant proteins like oleosin can be formulated more easily than antibodies and can be functionalized with precision through genetic engineering. We cloned S1-binding mini-protein genes called LCBx, previously designed by David Baker's laboratory (UW Seattle), to the N-terminus of oleosin, expressing Oleo-LCBx proteins in E. coli. These proteins largely formed 10-100 nm micelles as verified by dynamic light scattering. Two proteins, Oleo-LCB1 and Oleo-LCB3, were seen to completely and irreversibly block transduction by both wild-type and delta variant PVs into 293T-hsACE2 cells at 10 μM. Presented in multivalent micelles, these proteins reduced transduction by PVs down to a functional protein concentration of 5 nM. Additionally, Oleo-LCB1 micelles outperformed corresponding synthetic LCB1 mini-proteins in reducing transduction by PVs. Tunable aqueous solubility of recombinant oleosin allowed incorporation of peptides/mini-proteins at high concentrations within micelles, thus enhancing drug loading. To validate the potential multifunctionality of the micelles, we showed that certain combinations of Oleo-LCB1 and Oleo-LCB3 performed much better than the individual proteins at the same concentration. These micelles, which we showed to be non-toxic to human cells, are thus a promising step toward the design of modular, multifunctional therapeutics that could bind to and inactivate multiple receptors and proteins necessary for the infection of the SARS-CoV-2 virus.