Molecular Cloning, Cell-Surface Displayed Expression of VHH Against VEGF Expressed in E. Coli by Ice Nucleation Protein (INP)

Abstract

Angiogenesis plays a crucial role in various physiological and pathological processes, including tumor growth, where VEGF is considered the most critical factor in this process. Nowadays, the production of single-domain antibodies (VHH) with the ability to inhibit growth factors in cancer tumors is emerging as a novel approach in cancer therapy. In our previous research, it was identified that camelid VHHs generated through phage display against VEGF play a significant role in the inhibition of VEGF. Among these VHHs, the one with the highest affinity for the VEGF receptor was selected from the phage display VHH library. Considering the efficiency of surface expression in E. coli using the ice nucleation protein (INP) anchoring motif, this system was employed for protein expression. Given that only the C-terminal domain of the INP is involved in cell surface attachment, a gene structure of 537 bp starting from the InaK gene was utilized in the design. Surface expression was performed using the pET-21a containing INP and VEvhh10 against VEGF. The results indicated that the INP sequence is a suitable option for surface expression of VEvhh10 in E. coli. After the production of unbound VEvhh10, isolation, and purification by centrifugation and multiple washes were carried out, followed by examination of its binding to VEGF. The successful binding of VEvhh10 to VEGF demonstrates that the resulting non-fused protein could be utilized for future therapeutic and clinical diagnostic applications in patients.

Keywords

• bacterial surface display
• ice nucleation protein
• vhh
• angiogenesis
• vascular endothelial growth factor.