2026 Theses Doctoral

Engineering Selective and Functional Protein Encapsulation by Complex Coacervation

Ahn, So Yeon

Complex coacervation offers a powerful platform for protein encapsulation with applications ranging from protein purification to intracellular delivery. However, the polyampholytic character, folded structure, and charge regulation capacity of globular proteins cause their coacervation behavior to deviate substantially from ideal polymer models. This thesis investigates and exploits these deviations across three aims: understanding the selectivity of protein complex coacervation in multiprotein mixtures, engineering biosynthetic nanoparticles for defined structural assembly, and programming nanoparticle physicochemical properties for intracellular protein delivery.

In Chapter 2, a panel of spectrally separated fluorescent proteins mimicking the charge profile of the E. coli proteome was used to characterize protein phase behavior in multiprotein mixtures. Protein coacervation was found to synchronize in the mixture, and subtle differences in residue-level ionization state and charge regulation capacity became amplified into measurable differences in phase separation windows. This understanding was applied to selectively purify proteins of interest from the mixture with high purity and recovery.

In Chapter 3, biosynthetically produced multi-domain intrinsically disordered polypeptides (mdIDPs) comprising a neutral unstructured corona domain and a cationic histone-derived assembly domain were designed to self-assemble with anionic globular protein cargo into monodisperse complex coacervate core micelle nanoparticles. The cargo protein acts as an active assembly driver, enabling encapsulation efficiencies exceeding 90%. Rational design rules governing nanoparticle formation were established and demonstrated to be generalizable across a range of anionic globular protein cargos.

In Chapter 4, the mdIDP sequence was engineered to program nanoparticle properties for intracellular delivery. Aromatic substitutions (𝜋 variants) enhanced membrane affinity through favorable bilayer-water interface partitioning, while cysteine substitutions (C variants) provided physiological stability and redox-responsive cytosolic disassembly. Nanoparticle formation was identified as the critical driver of cellular uptake through multivalent membrane engagement. The optimized mdIDP variant with both strategies blended achieved optimal balance of stability in the physiological condition, membrane interaction, and cytosolic disassembly, confirmed by nuclear GFP localization as definitive evidence of cytosolic protein delivery.

Together, this thesis establishes protein sequence as the primary design parameter for engineering selective and functional protein encapsulation by complex coacervation.