Labeling Cell Surface Receptors with Ligand.BirA* Bispecifics

Mays Alwash; Jean Gariépy

doi:10.1021/acsptsci.1c00192

Labeling Cell Surface Receptors with Ligand.BirA* Bispecifics

ACS Pharmacol Transl Sci. 2022 Feb 1;5(2):62-69. doi: 10.1021/acsptsci.1c00192. eCollection 2022 Feb 11.

Authors

Mays Alwash^{1

2}, Jean Gariépy^{1

2

3}

Affiliations

¹ Sunnybrook Research Institute, 2075 Bayview Avenue, Toronto, Ontario M4N 3M5, Canada.
² Department of Pharmaceutical Sciences, University of Toronto, 144 College Street, Toronto, Ontario M5S 3M2, Canada.
³ Department of Medical Biophysics, University of Toronto, 101 College St Suite 15-701, Toronto, Ontario M5G 1L7, Canada.

Abstract

BirA*, a mutant form of the biotinylating enzyme BirA, can nonspecifically biotinylate ε-amino groups on lysines of proteins. Based on the promiscuous labeling nature of BirA*, plasmids expressing fusion constructs of BirA* to a given ligand have been used to transfect eukaryotic cells, leading to the biotinylation of intracellular proteins interacting or in close proximity to such Ligand.BirA* constructs. Mass spectrometry performed on the recovered biotinylated partners allows one to map intracellular protein interactors, a technique known as BioID. In contrast, the expression and purification of recombinant Ligand.BirA* constructs could serve as a powerful tool for labeling and detecting cell surface receptors. Here, we report the design and expression of recombinant Affibody.BirA* constructs, Z_EGFR:1907.BirA* and Z_HER2:243.BirA*, as protein bispecifics able to biotinylate their respective receptors EGFR and HER2 on the surface of MDA-MB-231 (EGFR⁺, EpCaM⁺, and CD44⁺) and SK-OV-3 (HER2⁺⁺, EGFR⁺, EpCaM⁺, and CD44⁺) cancer cells. These Affibody.BirA* constructs retain both their BirA* enzymatic activity as well as their receptor-binding function. Importantly, MDA-MB-231 and SK-OV-3 cells biotinylated with Affibody.BirA* constructs did label their receptors EGFR and HER2 but did not biotinylate irrelevant antigens such as EpCaM or CD44 present on the surface of both cell lines. Ligand.BirA* bispecifics may represent a promising class of agents to identify unknown receptors on cell surfaces.