Human Granulocyte-Macrophage Colony-Stimulating Factor Fused to Elastin-Like Polypeptides Assembles Biologically-Active Nanoparticles.

Abstract

Human granulocyte-macrophage colony-stimulating factor (hGMCSF) is crucial in the immune system as it stimulates survival, proliferation, differentiation, and functional activation of myeloid hematopoietic cells. hGMCSF is integral to approved therapies, including monoclonal antibodies against checkpoint inhibitors, chimeric antigen receptors, and prevention of chemotherapy-induced neutropenia. Recombinant hGMCSF can be purified from Escherichia. coli; however, it forms inclusion bodies that require solubilization and refolding. Alternatively, this manuscript describes its fusion with an elastin-like polypeptide (ELP). Previously reported as purification tags and solubility enhancers, ELPs are recombinant polypeptides that undergo reversible temperature-dependent phase separation. This report is the first to show that fusion to an ELP enables direct purification of hGMCSF fusions from the soluble fraction of bacterial lysate. Surprisingly, these ELP-fusions assemble stable, small, spherical nanoparticles that maintain pro-mitotic activity of hGMCSF. These nanoparticles exhibit ELP-mediated phase separation; however, nanoparticle assembly significantly increases the entropic and enthalpic cost of phase separation compared to ELP alone. The attachment of a high molecular weight ELP to a difficult-to-express protein, like hGMCSF, appears to be a useful strategy to stabilize bioactive, protein-based nanoparticles, which may have broad applications in medicine and biology.