Recombinant protein expression by mammalian cells is essential for evaluation in the development of drugs targeting proteins which are difficult to be obtained with conventional prokaryotic expression systems, such as membrane proteins, secreted proteins, or proteins with post-translational modifications. NMR analysis using proteins stable isotope-labeled by mammalian cells is less commonly performed because of the prohibitive cost and labor involved, however, it has the potential to provide information on the interaction between therapeutic targets and drug candidates in solution at atomic resolution, which cannot be obtained using other methods. In this study, we demonstrated stable isotope labeling of a therapeutic target by a mammalian cell expression system that can be used to identify the interaction interface of a target molecule with a middle-molecule inhibitor by solution NMR analysis.
We used the soluble neonatal Fc receptor (sFcRn), which functions in the heterodimeric form, as a model protein. sFcRn was prepared by transient expression in HEK293 cells and secretion outside the cells. We performed amino-acid (AA) selective labeling of sFcRn by replacing the selective natural AA in the culture media with the corresponding 13C- or 15N-labeled AA. sFcRn was successfully labeled with four types of AAs, one of which had not been reported previously. In addition, one type of AA required the addition of a single inhibitor of aminotransferase to improve the labeling efficiency enough to detect its NMR signal.
The labeled sFcRn was used for interaction analysis with SYN13271,2, a known cyclic peptide-type inhibitor. The chemical shift changes and intermolecular NOE showed that SYN1327 binds to the interaction interface with Fc. In contrast, SYN1327 induced little chemical shift changes in the residues outside the interaction interface with Fc. The site-specific interaction of sFcRn with SYN1327 may be responsible for the selective inhibition of the interaction between sFcRn and IgG.