Exploiting SUMO Fusion Technology for Enhanced Expression of Nanobodies Targeting Vascular Endothelial Growth Factor (VEGF) in Escherichia coli

Abstract

In recent years, nanobodies have emerged as invaluable tools in various research and therapeutic arenas owing to their compact size, robust stability, and high specificity. Notably, vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis, and plays a pivotal role in cancer and other pathological conditions. Consequently, the development of nanobodies targeting VEGF has garnered substantial attention. However, conventional expression methods for nanobodies in Escherichia coli often encounter challenges such as protein misfolding, low yields, and insolubility. In response to these hurdles, small ubiquitin-related modifier (SUMO) fusion technology has emerged as a promising strategy for enhancing the soluble production of nanobodies in E. coli. Here, we report the expression, purification, and characterization of VHH (variable domain of heavy chain antibodies) in an E. coli expression system using a (SUMO) fusion partner. The fusion gene (SUMO-active site TEV-VHH) was cloned into the pET-28(a+) expression vector and transferred into E. coli strain BL21 codon+. VEvhh10 expression was optimized using 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 22 hours at 25°C in Luria Broth (LB). Protein expression was observed in soluble form on 15% SDS-PAGE. The supernatant of SUMO-VEvh10 was collected and purified by Nickel-nitrilotriacetic acid (Ni-NTA) chromatography. The biological activity of VEvhh10 was assessed by examining its binding to VEGF. The successful binding of VEvhh10 to VEGF suggests that this non-fused protein could be utilized in future therapeutic and clinical diagnostic applications.

Keywords

• vhh (variable domain of heavy chain antibodies)
• vascular endothelial growth factor (vegf)
• sumo fusion technology