A simple, rapid, resin-free platform for purification of vascular endothelial growth factor using a calcium-responsive fusion protein

Abstract

Background

Vascular endothelial growth factor (VEGF) is widely used in regenerative medicine and therapeutic research. However, the purification of recombinant VEGF largely relies on affinity chromatography, which requires expensive chromatographic columns, specialized equipment, and multistep processing. These column-based workflows increase operational complexity and cost, particularly for large-scale production. Therefore, the development of an alternative purification strategy to conventional chromatography-based purification for VEGF is needed.

Findings

In this study, we developed a chromatography-free VEGF purification strategy using an anti-VEGF-scFv–calsequestrin (CSQ) fusion protein that enables calcium-dependent affinity precipitation. The fusion protein retained strong binding affinity for VEGF (K_d = 1.1 nM) while exhibiting rapid and reversible Ca²⁺-dependent polymerization. Upon CaCl₂ addition, the anti-VEGF-scFv-CSQ–VEGF complex rapidly formed aggregates, enabling efficient separation of VEGF from impurities. Using this strategy, VEGF was purified within 30 min with a purity of 94% and a yield of 93%. SEC-HPLC analysis confirmed a purity of 94.3%, and host cell protein contamination was reduced from 1.44 × 10⁴ ppm to 774 ppm. The fusion protein also maintained stable purification performance over five repeated cycles, with VEGF recovery consistently maintained above 85%.

Conclusions

These findings demonstrate that the scFv-CSQ fusion protein enables rapid separation of VEGF through calcium-dependent polymerization. This column-free mechanism reduces operational cost and technical complexity, highlighting its potential as an alternative to conventional chromatography-based purification.

Graphical abstract

Keywords

Vascular endothelial growth factor / Purification / Fusion protein / Calsequestrin / Single-chain variable fragment / Non-chromatographic purification

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Funding

National Research Foundation of Korea(RS-2023-00246123)

Startup Growth Technology Development Program(RS-2024-00441536)

Korea Institute of Ceramic Engineering and Technology(2410013749)

RIGHTS & PERMISSIONS

The Author(s)