Expression of deuterium-labeled NISTFab fragment in the methylotrophic yeast Komagataella phaffii for NMR studies

Published

October 31, 2023

Author(s)

Zvi Kelman, John Marino, William Brad O'Dell, Robert Brinson

Abstract

Deuterium isotope labeling is an essential tool in overcoming size limitations for molecules larger than 30 kilodaltons (kDa) in nuclear magnetic resonance (NMR) spectroscopy. The yeast-expressed antigen-binding fragment (Fab) of NIST RM 8671 NISTmAb, called yNISTFab, is a properly folded 50 kDa protein with native disulfide linkages when expressed in methylotrophic Komagataella phaffii (formerly Pichia pastoris). The production of deuterated yNISTFab is necessary to obtain well-resolved TROSY-HMQC NMR spectra for assignment of backbone amide chemical shifts. We examined growth conditions and effects of media composition to maximize biomass generation and expression of isotopically enriched 50 kDa Fab fragment. Triple-labeled yNISTFab with 93% deuteration reduced the 1H- and 15N-linewidths in the NMR spectra, allowing a sequence-specific assignment of amide crosspeaks - a key step toward structural and dynamic studies of Fab fragments and intact antibodies.

Citation

EPJ Web of Conferences

Pub Type

Journals

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Keywords

monoclonal antibodies, mAb, yeast expression, NMR, protein labeling

Citation

Kelman, Z. , Marino, J. , O'Dell, W. and Brinson, R. (2023), Expression of deuterium-labeled NISTFab fragment in the methylotrophic yeast Komagataella phaffii for NMR studies, EPJ Web of Conferences, [online], https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=936944 (Accessed October 9, 2025)