Generation and characterization of nanobodies targeting GPCR

Shenglan Zhang; Zhiran Fan; Jianfeng Liu

doi:10.52601/bpr.2023.230026

Generation and characterization of nanobodies targeting GPCR

Biophys Rep. 2024 Feb 29;10(1):22-30. doi: 10.52601/bpr.2023.230026.

Authors

Shenglan Zhang¹, Zhiran Fan¹, Jianfeng Liu^{1

2}

Affiliations

¹ Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), Guangzhou 510005, China.
² Cellular Signaling laboratory, International Research Center for Sensory Biology and Technology of MOST, Key Laboratory of Molecular Biophysics of MOE, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China.

Abstract

G protein-coupled receptors (GPCRs) are a large family of cell membrane proteins that are important targets for drug discovery. Nanobodies, also known as VHH (variable domains of heavy chain-only antibodies, HcAbs) antibodies, are small antibody fragments derived from camelids that have gained significant attention as potential therapeutics for targeting GPCRs due to their advantages over conventional antibodies. However, there are challenges in developing nanobodies targeting GPCRs, among which epitope accessibility is the most significant because the cell membrane partially shields the GPCR surface. We developed a universal protocol for making nanobodies targeting GPCRs using the cell membrane extract of GPCR-overexpressing HEK293 cells as the llama/alpaca immunization antigen. We constructed an immune VHH library and identified nanobodies by phage display bio-panning. The monoclonal nanobodies were recombinantly expressed in Escherichia coli (E. coli) and purified to characterize their binding potency.

Keywords: G protein-coupled receptor; GPCR; Heavy chain-only antibody; Nanobody; Single domain antibody.