Expressed Protein Ligation in Flow

The development of a flow chemistry platform for the generation of modified protein targets via expressed protein ligation (EPL) is described. The flow EPL platform enables efficient ligation reactions with high recoveries of target protein products and superior reaction rates compared to corresponding batch processes. The utility of the flow EPL technology was first demonstrated through the semisynthesis of the tick-derived chemokine-binding protein ACA-01 containing two tyrosine sulfate modifications. Full-length, sulfated ACA-01 could be efficiently assembled by ligating a recombinantly expressed C-terminal protein fragment and a synthetic sulfopeptide thioester in flow. Following folding, the semisynthetic sulfoprotein was shown to exhibit potent binding to a variety of pro-inflammatory chemokines. In a second modified protein target, we employed an in-line flow EPL-photodesulfurization strategy to generate both unmodified and phosphorylated forms of human β-synuclein by fusing a recombinant protein thioester, generated through cleavage of an intein fusion protein, and a synthetic (phospho)peptide. The semisynthetic proteins were assembled in 90 min in flow, a significant improvement over corresponding batch protein assembly, and enabled access to tens of milligrams of high purity material. Flow EPL has the potential to serve as a robust technology to streamline access to homogeneously modified proteins for a variety of applications in both academia, as well as in the pharmaceutical and biotechnology sector.