Better strategies to engineer rapid, reversible, and specific biological switches will improve disease models, interpretation of ‘-omics’ data, selection of drugs with specificity for target processes, and cellular therapies ranging from tissue engineering to chimeric antigen receptors. To develop a new synthetic biology toolkit, we have repurposed ‘protein-polymers’ derived from human tropoelastin. These elastin-like polypeptides (ELPs) can be fused at the gene level with functional proteins and expressed in cells. ELPs are repetitive peptides that reversibly phase separate without damaging host cells. Their phase behavior depends on variables, such as temperature, molecular weight, and concentration. We recently described how ELPs fused with cytosolic clathrin-light chain alter endocytosis and activity of G-coupled protein receptors in a temperature-dependent manner. Here we provide alternative evidence that ELPs can be expressed directly in fusion with the receptor tyrosine kinase known as the epidermal growth factor receptor(EGFR). The ELP-mediated phase separation induces EGFR internalization to early endosomes, phosphorylation of its receptor tyrosine kinase domain, and the downstream activation/phosphorylation of kinases ERK1/2 in HEK293T cells. This activity is similar to that achieved by addition of a soluble ligand, epidermal growth factor. Neither EGFR alone nor a temperature-insensitive EGFR-ELP control showed similar thermal modulation. EGFR activation can be tuned by selection of the ELP, occurs within minutes, and can be maintained over longer durations by temperature oscillation. Having demonstrated that transfected ELP fusions can modulate endocytosis and cell signaling, we next explored whether exogenous ELPs bound to cell-surface receptors can modulate temperature-dependent phase separation of cell surface receptors. A library of purified single-chain antibodies fused with ELPs was first bound to cell-surface receptors (CD19, CD20, CD3) involved with B and T cell maturation and lymphoma. When heated, these receptors undergo-temperature-dependent clustering that causes receptor internalization. We conclude that ELP fusions can be triggered by mild heating to assemble various targets inside the cell membrane, in the cytosol, or when bound to external cell-surface receptors. This synthetic biology toolkit is a powerful strategy to rapidly, specifically, and reversibly manipulate receptors important in biology and disease.

Intra- and extra-cellular manipulation of cell signaling using elastin-like polypeptides

253rd National Meeting (2017)

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