Creating a Selective Nanobody Against 3-Nitrotyrosine Containing Proteins

Elise M Van Fossen; Sonia Grutzius; Carl E Ruby; Dan V Mourich; Chris Cebra; Shay Bracha; P Andrew Karplus; Richard B Cooley; Ryan A Mehl

doi:10.3389/fchem.2022.835229

Creating a Selective Nanobody Against 3-Nitrotyrosine Containing Proteins

Front Chem. 2022 Feb 21:10:835229. doi: 10.3389/fchem.2022.835229. eCollection 2022.

Authors

Elise M Van Fossen¹, Sonia Grutzius¹, Carl E Ruby², Dan V Mourich², Chris Cebra², Shay Bracha³, P Andrew Karplus¹, Richard B Cooley¹, Ryan A Mehl¹

Affiliations

¹ Oregon State University, Department of Biochemistry and Biophysics, Agricultural and Life Sciences, Corvallis, OR, United States.
² Oregon State University, Department of Clinical Sciences, College of Veterinary Medicine, Corvallis, OR, United States.
³ Department of Small Animal Clinical Sciences (VSCS), Texas A&M College of Veterinary Medicine and Biomedical Sciences, College Station, TX, United States.

Abstract

A critical step in developing therapeutics for oxidative stress-related pathologies is the ability to determine which specific modified protein species are innocuous by-products of pathology and which are causative agents. To achieve this goal, technologies are needed that can identify, characterize and quantify oxidative post translational modifications (oxPTMs). Nanobodies (Nbs) represent exquisite tools for intracellular tracking of molecules due to their small size, stability and engineerability. Here, we demonstrate that it is possible to develop a selective Nb against an oxPTM protein, with the key advance being the use of genetic code expansion (GCE) to provide an efficient source of the large quantities of high-quality, homogenous and site-specific oxPTM-containing protein needed for the Nb selection process. In this proof-of-concept study, we produce a Nb selective for a 3-nitrotyrosine (nitroTyr) modified form of the 14-3-3 signaling protein with a lesser recognition of nitroTyr in other protein contexts. This advance opens the door to the GCE-facilitated development of other anti-PTM Nbs.

Keywords: genetic code expansion; nanobody; nitrotyrosine; oxidative post translational modification; single domain antibody.

Abstract

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