Enzymatically controlled release of proteins and peptides: A promising, alternative secretion approach.

The biological activity of many proteins is influenced by glycosylation, underscoring the essential role of the glycosylation process. However, no established recombinant technique currently enables the controlled shedding of glycosylated extracellular loops from transmembrane proteins. Here, we describe enzymatically controlled release of proteins and peptides (ENCOREP), a strategy that enables in situ expression at the cell surface and protease-mediated release, followed by collection from the cell culture medium. Using ENCOREP, we achieved the production of glycosylated peptides. These glycopeptides, derived from the large extracellular loop of the highly glycosylated CD63, illustrate the type of targets that are otherwise inaccessible with current methods but can be readily obtained using ENCOREP. Overall, ENCOREP provides a rapid and reliable approach to obtain glycosylated proteins or peptides while bypassing the conventional signal peptide-dependent secretory pathway.