Production of Artificially Doubly Glycosylated, ¹⁵N Labeled Prion Protein for NMR Studies Using a pH-Scanning...

Bacterially expressed proteins used in NMR studies lack glycans, and proteins from other organisms are neither ¹⁵N labeled nor glycosylated homogeneously. Here, we add two artificial glycans to uniformly ¹⁵N labeled prion protein using a buffer system that evolves over a pH range to accommodate the conflicting pH requirements of the substrate and enzymes without the need to fine-tune buffer conditions. NMR and CD spectroscopy of the protein indicates that the glycans do not influence its fold.