Bioorthogonal Functionalization of Elastin-like Polypeptides

Recombinant technology has given us the powerful ability to imagine and create novel biological entities, from potent therapeutics to functionally active materials. By harnessing nature’s building blocks and reconfiguring these components, recombinant engineering unlocks the potential to tailor drug specificity and pharmacokinetics, rationally design biomaterials, understand and define protein structure, and probe cellular function with molecular precision. These technological feats are made possible with a few simple biological ingredients: nucleotides, sugars, and amino acids. These components, exquisitely crafted by evolution, are individually combined in useful ratios and precise sequences in living systems to synthesize DNA, RNA, polysaccharides, and proteins. These macromolecules collectively support organismal structure and function and give rise to the incredible diversity in Charles Darwin’s “great tree” of life. However, the seemingly infinite potential for new materials built from these components is, in fact, limited. The chemical identity of these building blocks – with a particular focus herein on the twenty naturally-occurring amino acids – limits the scope and functionality of the recombinant materials we can produce. In order to functionalize these products, to fundamentally change their chemical identity while preserving their biological functionality, we require the finesse of bioorthogonal chemistries and modification techniques.

Bioorthogonal reactions modify biological materials within living systems without perturbing function, much as two orthogonal lines can extend in different directions and intersect only at a single point. That point of intersection can be precisely defined through recombinant technology and gives us access to new classes of biomaterials. The term “bioorthogonal”, coined by Carolyn Bertozzi, importantly defines these unique chemistries, which inertly co-exist with biology until the exact moment when the desired reactions are initiated, to enhance – and even transform – biological systems.

Bioorthogonal modification of proteins will, by definition, require expansion of the biochemical toolbox; there are a variety of techniques used to achieve this goal. In these studies, we explore the use of genetic code expansion for incorporation of unnatural amino acids. This technology permits co-translational incorporation of amino acids with unique and non-canonical R-groups directly into the polypeptide backbone of a protein or biopolymer. These residues introduce unique chemical reactivity for further functionalization with desired moieties or chemical transformation.

We have used this technology to develop novel therapeutic and material platforms comprised of a unique biopolymer, elastin-like polypeptide (ELP). This thermally responsive biopolymer is easily recombinantly synthesized, though more biochemically complex ELPs require successful bioorthogonal modification. We designed the unnatural amino acid-containing ELPs necessary to enable our strategies for developing three distinct biomaterial platforms: 1) photoreactive ELPs which can generate stable hydrogel particles spanning four orders of magnitude in size; 2) a universal strategy for drug-loaded, targeted ELP nanoparticles by incorporation of a unique site for drug attachment; 3) a sustained-release therapeutic for treatment of brain tumors combining proteins of distinct cellular origin.

We have combined existing tools, technologies, and materials to generate these novel platforms with utility in biomaterials, drug delivery, and cancer therapeutics. The optimizations performed in developing each of these systems will inform future studies with similar goals; similarly, the reactions and strategies employed will contribute to furthering our understanding of the full potential of these important bioorthogonal chemistries.