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.

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 1H-15N 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.