Intracellular phase separation of globular proteins facilitated by short cationic peptides.

Phase separation provides intracellular organization and underlies a variety of cellular processes. These biomolecular condensates exhibit distinct physical and material properties. Current strategies for engineering condensate formation include using intrinsically disordered domains and altering protein surface charge by chemical supercharging or site-specific mutagenesis. We propose adding to this toolbox designer peptide tags that provide several potential advantages for engineering protein phase separation in bacteria. Herein, we demonstrate the use of short cationic peptide tags for sequestration of proteins of interest into bacterial condensates and provide a foundational study for their development as tools for condensate engineering. Using a panel of GFP variants, we demonstrate how cationic tag and globular domain charge contribute to intracellular phase separation in E. coli and observe that the tag can affect condensate disassembly at a given net charge near the phase separation boundary. We showcase the broad applicability of these tags by appending them onto enzymes and demonstrating that the sequestered enzymes remain catalytically active.

Self-assembly of globular proteins with intrinsically disordered protein polyelectrolytes and block copolymers.

Intrinsically disordered polypeptides are a versatile class of materials, combining the biocompatibility of peptides with the disordered structure and diverse phase behaviors of synthetic polymers. Synthetic polyelectrolytes are capable of complex phase behavior when mixed with oppositely charged polyelectrolytes, facilitating nanoparticle formation and bulk phase separation. However, there has been limited exploration of intrinsically disordered protein polyelectrolytes as potential bio-based replacements for synthetic polyelectrolytes. Here, we produce negatively charged, intrinsically disordered polypeptides, capable of high-yield expression in E. coli and use this intrinsically disordered peptide to produce entirely protein-based polyelectrolyte complexes. The complexes display rich phase behavior, showing sensitivity to charge density, salt concentration, temperature, and charge fraction. We characterize this behavior through a combination of turbidity assays, dynamic light scattering, and transmission electron microscopy. The robust expression profile and stimuli-responsive phase behavior of the intrinsically disordered peptides demonstrates their potential as easily producible, biocompatible substitutes for synthetic polyelectrolytes.

Genetic and Covalent Protein Modification Strategies to Facilitate Intracellular Delivery.

Protein-based therapeutics represent a rapidly growing segment of approved disease treatments. Successful intracellular delivery of proteins is an important precondition for expanded in vivo and in vitro applications of protein therapeutics. Direct modification of proteins and peptides for improved cytosolic translocation are a promising method of increasing delivery efficiency and expanding the viability of intracellular protein therapeutics. In this Review, we present recent advances in both synthetic and genetic protein modifications for intracellular delivery. Active endocytosis-based and passive internalization pathways are discussed, followed by a review of modification methods for improved cytosolic delivery. After establishing how proteins can be modified, general strategies for facilitating intracellular delivery, such as chemical supercharging or inclusion of cell-penetrating motifs, are covered. We then outline protein modifications that promote endosomal escape. We finally examine the delivery of two potential classes of therapeutic proteins, antibodies and associated antibody fragments, and gene editing proteins, such as cas9.

Macro- and Microphase Separated Protein-Polyelectrolyte Complexes: Design Parameters and Current Progress.

Protein-containing polyelectrolyte complexes (PECs) are a diverse class of materials, composed of two or more oppositely charged polyelectrolytes that condense and phase separate near overall charge neutrality. Such phase-separation can take on a variety of morphologies from macrophase separated liquid condensates, to solid precipitates, to monodispersed spherical micelles. In this review, we present an overview of recent advances in protein-containing PECs, with an overall goal of defining relevant design parameters for macro- and microphase separated PECs. For both classes of PECs, the influence of protein characteristics, such as surface charge and patchiness, co-polyelectrolyte characteristics, such as charge density and structure, and overall solution characteristics, such as salt concentration and pH, are considered. After overall design features are established, potential applications in food processing, biosensing, drug delivery, and protein purification are discussed and recent characterization techniques for protein-containing PECs are highlighted.