Metabolic ¹³C-Labeling of IgA Fc Fragment by Transient Mammalian Expression for NMR Analysis

The atomic description of glycosylated proteins is essential for gaining an understanding of the physiological roles of protein glycosylation. X-ray crystallographic and cryo-EM analyses currently play a central role in the 3D structural determination of biological macromolecules. 3D structural information of glycosylated proteins remains limited due to the weak or absent electron densities of the glycan parts. Solution nuclear magnetic resonance (NMR) analysis is unique in that the conformation and dynamics of the glycan part can be obtained experimentally at atomic resolution. The sensitivity of NMR can be greatly enhanced through ¹³C isotope labeling of the glycoprotein glycan, and such labeling provides a new dimension in the NMR spectra. Specifically, signal assignment of the glycoprotein glycan can be attained unambiguously through scalar and nuclear Overhauser effect (NOE) connectivities. To this end, efficient and cost-effective ¹³C-labeling methodology is needed, through good yields in glycoprotein expression and purification. Here we introduce our method for the preparation of a ¹³C-labeled immunoglobulin A (IgA) Fc fragment using a transient mammalian expression system.