Protein therapeutics play a significant role in medical advancements, however, intracellular uptake of protein-based therapeutics remain a challenge. The majority of developed protein therapeutics do not readily cross cell membranes, but rather act primarily on extracellular or membrane-bound targets. Thus, intracellular protein delivery methods must be developed to realize the full therapeutic potential of exogenous protein therapeutics. To allow for cellular uptake, liquid peptide-based droplets can be designed by mimicking the physicochemical properties of endogenous membraneless organelles. Droplet formation occurs by complex coacervation, the liquid-liquid phase separation of oppositely charged polyelectrolytes, to achieve mesoscale self-assembly. We biosynthesized block copolypeptides with a net neutral block and a positively charged block, to assemble polyelectrolyte complex (PEC) micelles when combined with model anionic globular protein cargo. The two components spontaneously form uniform nano-sized micelles when mixed and are held together by non-covalent interactions that allow the condensation of the cargo protein while ensuring its solubility in the liquid phase. Here, we describe the design of biosynthetic PEC micelles that encapsulate anionic GFP as a model cargo protein by complexing with a net neutral fluorescent protein mScarlet-I fused to the disordered region of Histone H5, a highly charge dense cationic protein. Optimal parameters for stable micelle formation were systematically studied using genetic modification to vary protein net charge and charge density. The engineered block copolypeptides were mixed with anionic cargo protein at varying mixing ratios to promote assembly. The phase behavior of engineered proteins were evaluated via microscopy, dynamic light scattering, and turbidity assays to assess stable micelle formation.
