Development of a Long-Acting Myeloid-Derived Growth Factor via Site-Specific PEGylation

Extracellular myeloid-derived growth factor (MYDGF) can improve organ repair. However, short <i>in vivo</i> half-life hampers its therapeutic application. Herein, we developed a long-acting MYDGF via site-specific PEGylation at its C-terminus. Bacterially overexpressed human MYDGF carrying a C-terminal Asn-Ala-Leu tripeptide motif was first ligated with a synthetic azido-functionalized Gly-Ile-Gly-Lys(N3) tetrapeptide linker via catalysis of [G238 V]BmAEP1, an engineered bamboo-derived asparaginyl endopeptidase (AEP)-type peptide ligase. Thereafter, the azido-functionalized MYDGF was efficiently conjugated with a commercially available dibenzocyclooctyne (DBCO)-functionalized linear PEG30000 via copper-free click chemistry. The site-specifically PEGylated MYDGF (PEG-MYDGF) retained high <i>in vitro</i> activity and showed a much longer <i>in vivo</i> half-life in mice compared with unmodified MYDGF. In diabetic mice, PEG-MYDGF significantly promoted wound healing after subcutaneous injection. Thus, PEG-MYDGF represents a long-acting biologic with therapeutic potential. The present enzymatic peptide ligation and copper-free click chemistry-based approach could be applied to other proteins for site-specific conjugation with various functional moieties.