Deglycosylation of VEGF Produced in Pichia pastoris Reduces its in Vitro Binding and Angiogenic Effect

Vascular endothelial growth factor (VEGF) is a key pro-angiogenic glycoprotein used in both research and therapeutic applications. While Pichia pastoris (Komagataella phaffii) provides a cost-effective expression system for recombinant VEGF, its glycosylation patterns differ significantly from those in mammalian systems, and the impact of these modifications remains underexplored. Recombinant human VEGF165 was expressed in P. pastoris, purified via heparin affinity chromatography, and enzymatically deglycosylated using Endo H. Both glycosylated (yVEGF_g) and deglycosylated (yVEGF_dg) forms were characterized for their binding to an anti-VEGF antibody, bevacizumab, via ELISA and SPR and evaluated for biological activity using HUVEC proliferation assays. Comparative analyses were also conducted with mammalian-expressed VEGF. Deglycosylated VEGF exhibited reduced binding affinity to bevacizumab and a lower proliferative effect on endothelial cells compared to its glycosylated counterpart. Despite the N-glycosylation site being structurally distant from receptor/antibody epitopes, glycosylation enhanced VEGF's in vitro binding kinetics and angiogenic activity. Partially glycosylated VEGF from P. pastoris even surpassed mammalian-expressed VEGF in inducing endothelial proliferation. These findings demonstrate that glycosylation significantly enhances the functional properties of recombinant VEGF produced in P. pastoris, emphasizing the importance of glycosylation control for the development of bioactive VEGF.