Backbone NMR assignment of the yeast expressed Fab fragment of the NISTmAb reference antibody

Strategies for the production of isotopically labelled Fab fragments of therapeutic antibodies in Komagataella phaffii (Pichia pastoris) and Escherichia coli for NMR studies

“…However, preparation of a 2 H- 15 N-NISTmAb-Fab sample resulted in a significantly lower yield due to the difficulty of yeast cells to grow to high cell densities. For this reason, Brinson and co-workers elected to use a triply labelled rich growth media to attenuate this problem in order to improve protein yield in the production of 2 H- 13 C- 15 N-NISTmAb-Fab for resonance assignment [ 34 ]. In contrast, expression of a single chain version of Fab fragments in the form of inclusion bodies in E. coli was much simpler and more efficient.…”

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

“…They both share identical primary sequences in their constant heavy 1 (C H 1) and constant light domain (C L ). In order to further validate our assignment, we compared it to the resonance assignment of backbone atoms of the NISTmAb-Fab (Solomon et al, 2023). It is expected that resonances arising from amides with the same local magnetic environment will have the same chemical shifts while others that have similar or slightly different environments will produce slightly or signi cantly different chemical shifts.…”

“…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.…”

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

“…Previous work has established that the constituent Fc and Fab domains retain the HOS of the parent molecule with only minor chemical shift perturbations observed for residues near in the hinge region [11]. Until our recent publication of NMR assignments of a Fab domain [12], only assignments of the Fc domain were available including from glycosylated and aglycosylated versions from CHO (Chinese Hamster Ovary) and Escherichia coli expression systems [11,13]. The first step in chemical shift assignment is to conduct a suite of triple-resonance NMR experiments to correlate the chemical shifts of the amide proton (HN) with the chemical shifts of Cα and Cβ [14].…”

“…We therefore further investigated K. phaffii culture growth conditions and purification protocols to achieve production of ~6.6 mg/L of triple-labeled yNIST-Fab with 93% deuteration. The TROSY-based spectra of this deuterated yNIST-Fab were acquired to demonstrate the suitability of the material for NMR experiments and enabled the first report of backbone assignments for a Fab domain [12]. 13 C, 15 N-ISOGRO ® (MilliporeSigma DCN-IG 608297-125)…”