文献类型：期刊文献

英文题名：Production of isotope-labeled nanobody KN035 in Pichia pastoris yields native-like conformation and PD-L1 binding activity comparable to KN035 expressed in mammalian cells

作者：Ren, Piaoran[1];Jiang, Mengzhen[1];Wang, Shenlin[1]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China

年份：2026

卷号：6

期号：3

外文期刊名：MAGNETIC RESONANCE LETTERS

收录：EI(收录号：20262420880056);WOS:【ESCI(收录号:WOS:001803570300001)】；

基金：NMR experiments for KN035 expressed in E. coli and P. pastoris were conducted on the 600 MHz spectrometer at the NMR Facility of the National Center for Protein Science (Shanghai). Experiments for the HEK293-expressed KN035 were performed on the 800 MHz spectrometer at East China University of Science and Technology. The work was supported by the National Key R&D Program of China (2024YFA0917100), the National Natural Science Foundation of China (22274050), the Shanghai Science and Technology Commission (contract numbers: 23J21900300, 24HC2810700), and the Fundamental Research Funds for the Central Universities. We also thank the staff members of the Nuclear Magnetic Resonance System at the National Facility for Protein Science in Shanghai (NFPS), Shanghai Advanced Research Institute, Chinese Academy of Sciences, China, for providing technical support and assistance in data collection and analysis.

语种：英文

外文关键词：Pichia pastoris; Nanobody; Isotope labeling; Nuclear magnetic resonance; Protein expression

摘要：Stable isotope-labeled proteins are indispensable for NMR-based structural studies. E. coli is a cost-effective and high-yield expression host, but it often fails to support proper folding and post-translational modifications required for functional human proteins. While mammalian cell systems produce correctly folded and modified proteins, their high cost limits widespread use. The methylotrophic yeast Pichia pastoris (P. pastoris) presents a compelling alternative as a single-celled eukaryotic host that combines advantages of simple and cost-effective microbial cultivation and eukaryotic protein processing capabilities. In this study, we systematically compared the spectra and ligand-binding activity of the VHH domain of therapeutic nanobody KN035, expressed in E. coli, P. pastoris, and HEK293 cells to discuss the feasibility of using P. pastoris to produce labeled nanobody for NMR studies. KN035 produced in P. pastoris and mammalian HEK293 cells displayed nearly identical 1 H- 15 N HSQC spectra, indicating highly similar folding. In contrast, KN035 expressed in E. coli formed inclusion bodies and, after refolding, exhibited conformational heterogeneity with largely different spectral patterns with HEK293 expressed KN035. NMR titration experiments with PD-L1 further revealed that P. pastoris-expressed KN035 bound its target with a mode indistinguishable from the HEK293-produced protein, while the E. coli-expressed sample showed altered spectra of the binding complex. Our results concluded that P. pastoris, with its simple single-cell culture requirements, can produce structurally similar and functionally active KN035, simulating mammalian cells production. This work establishes single-celled P. pastoris as a practical host for producing isotope-labeled nanobodies, combining the ease of microbial cultivation with the ability to generate properly folded eukaryotic proteins for structural and interaction studies. (c) 2026 Publishing services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).