O‐GlcNAc Engineering on a Target Protein in Cells with Nanobody‐OGT and Nanobody‐splitOGA

The monosaccharide O‐linked N‐acetyl glucosamine (O‐GlcNAc) is an essential and dynamic post‐translational modification (PTM) that decorates thousands of nucleocytoplasmic proteins. Interrogating the role of O‐GlcNAc on a target protein is crucial yet challenging to perform in cells. We recently reported a pair of methods to selectively install or remove O‐GlcNAc on a target protein in cells using an engineered O‐GlcNAc transferase (OGT) or split O‐GlcNAcase (OGA) fused to a nanobody. Target protein O‐GlcNAcylation and de‐O‐GlcNAcylation complements methods to interrogate the role of O‐GlcNAc on a global scale or at individual glycosites. Herein, we describe a protocol for utilizing the nanobody‐OGT and nanobody‐splitOGA systems to screen for O‐GlcNAc functionality on a target protein. We additionally include associated protocols for the detection of O‐GlcNAc and cloning procedures to adapt the method for the user's target protein of interest. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Target protein O‐GlcNAcylation of JunB using nanobody‐OGT Basic Protocol 2: Target protein deglycosylation of Nup62 using nanobody‐splitOGA Alternate Protocol: Verification of the O‐GlcNAc state of a tagged target protein through chemoenzymatic labeling Support Protocol: Cloning of new nanobody‐OGT/nanobody‐splitOGA and target protein pairs