Triggered sorting and co-assembly of genetically engineered protein microdomains in the cytoplasm.

The various highly compartmentalized, membrane-bound organelles are essential for cellular metabolism in eukaryotic cells.[1,2] The specialized composition of proteins and lipids in different organelles enables them to accomplish specialized processes, and sophisticated sorting mechanisms direct molecules to specific intracellular locations to maintain cellular functions.[2–4] Although it is ubiquitous to life that cells routinely generate and sort nanostructures, including protein complexes, organelles and chromosomes, our ability to similarly engineer and sort synthetic organelles in vivo remains primitive.[2,5,6] To address this challenge, this communication describes an innovative approach for co-assembly and self-sorting of materials inside living cells using genetically-encoded protein polymers – elastin-like polypeptides (ELPs).[7] ELPs are repetitive polypeptides with the sequence of (Val-Pro-Gly-Xaa-Gly)n derived from human tropoelastin, where Xaa and n represent the ‘guest residue’ identity and number of repeat units, respectively.[8,9] ELPs mediate self-assembly by temperature-triggered phase separation above their transition temperature (Tt).[10] Depending on the composition and arrangement of guest residues, monoblock ELPs form micro-structures distinct from diblock ELPs after assembly.[11–13] While monoblock ELPs form large protein coacervates, amphiphilic ELP diblock copolymers sometimes assemble nanoparticles of less than 100 nm in diameter.[14,15] Prior studies revealed that ELP fusion proteins assemble genetically engineered protein microdomains (GEPMs) in living eukaryotic or E. coli cells;[16,17] however, this communication is the first report exploring how ELPs can sort or assemble two distinct proteins. The hypothesis is that different monoblock ELPs with similar transition temperatures may spatially coassemble into mixed GEPMs, and that these will spatially sort from ELP diblock copolymers (Scheme 1). To test this hypothesis, three different monoblock ELPs and one diblock ELP were biosynthesized and purified, and their micro-structures were identified and characterized. The capability of these ELPs to spatially co-assemble and self-sort was evaluated using confocal laser scanning microscopy both in vitro and in the eukaryotic cytosol. These findings reveal a potentially powerful strategy for intracellular co-assembly and sorting of GEPMs, and may have utility in organizing synthetic organelles enriched in distinct functional proteins.