The development, use, and optimization of sortase-tag expressed protein ligation

Molecular imaging is an emerging field that seeks to combine the mechanistic detail of biochemical assays with the the broad phenotypic data obtained from non-invasive medical imaging. A cornerstone of molecular imaging is the ability to chemically attach a contrast-inducing payload to a targeting ligand, a protein that binds tightly and specifically to the targeted cell population. There are currently many approaches to bioconjugation. Chemical approaches have broad applicability, but lack the site-specificity and efficiency desired for contrast agent generation. Enzymatic techniques are quantitative, efficient, and site-specific, but often have limited scope and can add significant bulk to the targeting ligand or require additional downstream purification steps. In this work, we create a novel bioconjugation and protein purification technique, Sortase-Tag Expressed Protein Ligation (STEPL). By fusing the active domain of S. aureus Sortase A to the targeting ligand as part of a greater chimeric protein, we are able to combine protein purification and the C-terminal bioconjugation of that protein into a single step. Using a mass-action kinetics model, we prove that this results in a modular, efficient system capable of producing high-purity conjugated protein. We then use the system to generate contrast agents for fluorescent and magnetic imaging studies. The modularity of the STEPL system allows us to create an array of monospecific and bispecific targeting ligand dimers, linked by two different spacer regions. The dimers\u27 binding properties are probed and they are used to successfully bind and label cells, with the bispecific proteins enhancing contrast of a cell line that is positive for both targets. Finally the STEPL system is subjected to a number of directed evolution approaches and screened for a clone that could further improve STEPL\u27s efficiency and yield