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

## TL;DR

This paper introduces ENCOREP, a new method to release glycosylated proteins from cells, enabling the production of hard-to-obtain glycopeptides for research.

## Contribution

ENCOREP offers a novel, enzyme-driven approach to release glycosylated proteins from the cell surface, bypassing traditional secretion methods.

## Key findings

- ENCOREP successfully produced glycosylated peptides from the CD63 protein.
- The method uses an engineered protease for continuous release of extracellular loops.
- Released proteins are suitable for functional and biochemical assays.

## Abstract

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.

•ENCOREP releases glycosylated ectodomains from the cell surface into medium•Engineered tobacco etch virus protease drives continuous release•Applicable to GPCR extracellular loops and diverse glycosylated membrane proteins•Released proteins are ready for functional, biochemical, and binding assays

ENCOREP releases glycosylated ectodomains from the cell surface into medium

Engineered tobacco etch virus protease drives continuous release

Applicable to GPCR extracellular loops and diverse glycosylated membrane proteins

Released proteins are ready for functional, biochemical, and binding assays

Biochemistry; Protein; Cell biology