Cell-surface displayed expression of single domain antibody against VEGF in E. coli using N-terminal domain of ice nucleation protein (INP)

Alhafyan, Salim; Rezaei, Zeynab; Shahangian, S.S.; H. Sajedi, Reza

Cell-surface displayed expression of single domain antibody against VEGF in E. coli using N-terminal domain of ice nucleation protein (INP)

Document Type : Original Research

Authors

Salim Alhafyan ¹

Zeynab Rezaei ¹

S.S. Shahangian ²

Reza H. Sajedi ¹

¹ Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

² Department of Biology, Faculty of Sciences, University of Guilan, Rasht, Iran

Abstract

Angiogenesis is involved in many physiological and pathological processes, including tumor growth, and VEGF is considered as the most important factor in this process. Nowadays, the production of single-domain antibodies (VHH) with the characteristic of inhibiting growth factors in cancer tumors is one of the new strategies for cancer treatment. In the previous research, it was found that camel VHHs isolated from phage display against VEGF play an essential role in inhibiting it. Here, the VHH that had the highest affinity for the VEGF was selected. Ice nucleation protein (INP) was used as anchoring motif for surface expression of E. coli, owing to the efficiency of its N terminal domain, this system is used to express the VHH. Accordingly, a construct harboring the first 537 nucleotide of InaK gene and nucleotide fragments of TEV protease recognition site and VEvhh10 was designed to express this protein in the surface display of E. coli cells. The results showed that the INP anchor is a suitable candidate for promoting the surface expression of VEvhh10 in E. coli. After expression of VEvhh10, isolation and purification were performed using centrifugation and washing, and its binding to VEGF was investigated. The results showed that VEvhh10 successful bind to VEGF and it can be used for therapeutic applications and clinical diagnosis of patients in the future.

Keywords

Bacterial Surface Display

Ice Nucleation Protein (INP)

VHH

Vascular Endothelial Growth Factors (VEGF)

Subjects

Molecular biotechnology

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