Login / Signup

A robust antibody discovery platform for difficult-to-express G protein-coupled receptors.

Nam Hyuk KimSumin KangGa Hyeon ParkGoeun ShimTae Hyun KangYeon Gyu Yu
Published in: Protein science : a publication of the Protein Society (2022)
G protein-coupled receptors (GPCRs) are in the spotlight as drug targets due to the fact that multiple research results have verified the correlation between the activation of GPCRs and disease indications. This is because the GPCRs are present across the cell membranes, which interact with either extracellular ligands or other types of compartments, and simultaneously mediate intracellular signaling. Despite the importance of the GPCRs as drug targets, they are too difficult to express in soluble forms. Currently, the difficulty of preparing functional GPCRs and the lack of efficient antibody screening methods is the most challenging step in the discovery of antibodies targeting GPCRs. In this study, we developed a powerful platform that facilitates isolating GPCR-specific antibodies by obviating difficulties in GPCR preparation. The strategies include i) conjugation of P9 peptide, an envelope protein of Pseudomonas phi6, to the N-terminus of GPCRs to improve the expression level of the GPCRs in Escherichia coli (E. coli), ii) stabilization of the GPCRs in their active forms with amphiphilic poly-γ-glutamate (APG) to shield the seven hydrophobic transmembrane domains, and iii) further limiting the size of the APG complex to improve the chance to isolate antibodies targeting the proteins-of-interest. Capitalizing on the above strategies, we could prepare GPCR proteins in their active forms as facile as other general soluble antigen proteins. Furthermore, this protocol was validated to be successful in discovering three individual GPCR-specific antibodies targeting Glucagon-like peptide-1 receptor (GLP1R), C-X-C chemokine receptor type 4 (CXCR4), and Prostaglandin E2 receptor 4 (EP4) in this study. This article is protected by copyright. All rights reserved.
Keyphrases
  • escherichia coli
  • high throughput
  • small molecule
  • single cell
  • drug delivery
  • stem cells
  • ionic liquid
  • cystic fibrosis
  • mass spectrometry
  • amino acid
  • klebsiella pneumoniae