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

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.