Expression of ²H, ¹³C, ¹⁵N-labeled NIST-Fab fragment in the methylotrophic yeast Komagataella phaffii for nuclear magnetic resonance studies

Abstract

Labeling of proteins with deuterium is an essential tool in overcoming size limitations in the application of nuclear magnetic resonance (NMR) spectroscopy to proteins larger than 30 kilodaltons (kDa). A non-originator antigen-binding fragment (Fab) of NIST RM 8671 NISTmAb, so called yNIST-Fab, is a ~ 50 kDa protein, with 5 native disulfide linkages, that can be expressed in properly folded form in methylotrophic Komagataella phaffii (formerly Pichia pastoris). Further, the K. phaffii host can support the production of perdeuterated yNIST-Fab which is necessary to obtain well-resolved TROSY-based tripleresonance NMR spectra for chemical shift assignment of the peptide backbone resonances. Here, we examined growth conditions and effects of media composition to maximize biomass generation and expression yield of the 2H, 13C, 15N-enriched NIST-Fab fragment. Triple-labeled yNIST-Fab with ~93% deuteration reduced the 1HN, 15N and 13C-linewidths in the NMR spectra, allowing sequential NMR assignment of backbone resonance a key step toward sequence-specific structural and dynamic studies of Fab fragments and intact antibodies.

Backbone and methyl side-chain resonance assignments of the single chain Fab fragment of trastuzumab

“…Recently, that limit has been crossed. Solomon and coworkers reported the backbone resonance assignment of yeast produced NIST-mAb Fab (Chao et al, 2023;Solomon et al, 2023). However, more complex labelling schemes with yeast, such as methyl labelling, are not straightforward and thus require further developments.…”