Leveraging Intrinsically Disordered Polypeptides for Biotechnology Applications

Abstract

As the sequence space for proteins has continued to expand and new functions are discovered within cells, many of the preconceptions surrounding protein structure and function are slowly being shed. Intrinsically disordered regions within proteins have continued to step into the protein engineering spotlight as cellular mechanisms that rely on these unstructured domains are uncovered and biomolecular engineers begin to leverage their native functions for novel biotechnology applications. In this work, we demonstrated an application of a naturally inspired intrinsically disordered polypeptide – elastin-like polypeptide (ELP) – and de novo synthetic intrinsically disordered polypeptides (SynIDPs) towards two different areas of interest in biotechnology – drug delivery and synthetic biology. First, we explored the use intrinsically disordered polypeptides (IDPs) for improving the delivery of IL-12, a pro-inflammatory cytokine that suffers from a short half-life and narrow therapeutic window (Chapter 2). Our work sought to address these limitations through utilizing an ELP platform , which can undergo a lower critical solution temperature phase transition to a gel-like depot upon injection in vivo, to evaluate the individual and combinatorial effects of spatial delivery and sustained release of IL-12. Through a sortase-mediated transpeptidation between IL-12 and an ELP, we created an IL-12-ELP fusion that can be injected intratumorally or subcutaneously to form a sustained-release depot. In a B16F10 murine model, intratumoral injection of a depot-forming IL-12-ELP fusion significantly improved survival in mice compared to free IL-12 delivered intravenously or intratumorally. IL-12-ELP was retained within the tumor ~ 4-fold longer than free IL-12, resulting in higher CD8+ T cell recruitment at the tumor and local concentrations of inflammatory cytokines MCP-1, TNF-a, and IFN-γ at Day 2. We then explored a novel method of generating a library of synthetic peptides that were derived from surveying the sequence space of PGX1X2X3X4 motifs to generate de novo solubility tags (Chapter 3). A DNA library of 1020 unique gene sequences was pooled and amplified using rolling circle amplification prior to selecting for desired size fragments and ligating into a modified pET24 plasmid for bacterial expression. The library of SynIDPs was electroporated into bacteria and colonies harboring soluble SynIDPs were screened via colony filtration. Selected SynIDPs were further examined for their ratio of soluble to insoluble protein expression using western blot analysis, and candidates with the highest soluble expression were selected for further exploration. We characterized the top three highly soluble SynIDPs through MALDI-TOF, CD, and SAXS to characterize their size, secondary structure, and conformational flexibility. Lastly, we demonstrated the utility of the three highly soluble IDPs by fusing them to a set of three known inclusion body forming proteins – mTdT, TEV protease, and Z2-LO10 – that are commonly used enzymes in biotechnology applications or promising therapeutics in clinical trials. Upon fusion with any of the three SynIDPs, all three of the highly insoluble proteins demonstrated notable increases in soluble expression, as well as minimal loss of activity when fused. Finally, we applied both IDPs used in Chapters 2 and 3 towards improving the delivery of IL-15, a promising cytokine for cancer immunotherapy that suffers from a rapid clearance in vivo (Chapter 4). Through refolding IL-15 fused with an ELP, we demonstrated that we were able to generate an active and thermally responsive variant of IL-15 that could potentially sustain release of the molecule beyond its short half-life of 2.5 hours. Additionally, we demonstrated that using a longer version of a SynIDP derived from the previous chapter circumvents the need for refolding IL-15 when expressed in SHuffle E. coli while still maintaining activity and overcoming glomerular filtration size limits. Taken together, this body of work demonstrates the versatility that intrinsically disordered polypeptides can play in enhancing biotechnology applications. By combining existing tools derived from nature and generating novel platforms, we have demonstrated optimizations in improving drug delivery and protein solubility, which can help further advancements in therapeutics and synthetic biology.