Xing, Y.; Li, Q.; Campbell, P. G.; Ren, X.

In native tissue environments, growth factors (GFs) are often physically associated with the extracellular matrix (ECM) framework. Despite the enormous potential of the chemoselective click chemistry for GF functionalization of biomaterials to recapitulate such GF-ECM association, its application is limited by the lack of a universal strategy for reliable production of clickable GFs. Here we present a novel platform technology that leverages intrinsic post-translational protein glycosylation to enable site-specific metabolic engineering of GFs with azido tags for subsequent ECM hydrogel conjugation. Using Vascular Endothelial Growth Factor as a model, we demonstrated efficient, glycosylation-dependent azido incorporation during its recombinant expression with preserved bioactivity. We further expanded the utility of this strategy to non-glycosylated proteins through engineered N-linked glycosylation via the incorporation of a signal peptide that directs newly synthesized proteins to the secretory pathway where glycosylation takes place along with a sequon for glycan attachment. The resulting GF with site-specific azido incorporation can be effectively immobilized within dibenzocyclooctyne-bearing ECM hydrogel via the copper-free click chemistry, exhibiting sustained GF retention and delivering augmented angiogenic responses. Our approach thereby offers an unprecedented opportunity to streamline recombinant protein engineering for biomaterial functionalization in tissue engineering and regenerative medicine applications.