Production of high-affinity glycosylated anti-mouse conjugated nanobodies in Pichia pastoris

Nanobodies (NBs) are small antibody fragments derived from camelid heavy-chain antibodies which represent the minimal functional domain capable of antigen recognition and binding. NB are ten times smaller than conventional antibodies, exhibit a compact structure and high stability, making them ideal for recombinant production. The eukaryotic unicellular system Pichia pastoris provides multiple advantages for protein expression, including the ability to perform several eukaryotic posttranslational modifications. In this work, we engineered a modular plasmid sequence that, through specific restriction enzyme cuts and ligations, codes the expression of a secreted anti-mouse kappa chain NB fused with various accessory peptides in P. pastoris . This system enables the incorporation of a plastic binding sequence, a histidine tag (Hisx6) for purification, the horseradish peroxidase (HRP) enzyme for chemiluminescence detection, or the biotinylatable AviTag sequence, in multiple combinations. We successfully expressed and purified anti- kappa NBs fused to a Hisx6-tag (κNB) and to HRP -Hisx6-tag (κNB-HRP), with subsequent structural and functional characterization revealing high affinity for mouse immunoglobulins. The κNB- kappa light chain domain complex was modeled showing a fitted surface interaction of CDR3 domain. The position of a glycan in the complex was modeled predicting that glycan addition would not affect the interaction surface. Accordingly, no functional differences were observed in κNB after deglycosylation, indicating that high mannose glycan addition has not interfered with its binding capability. Moreover, glycosylated κNB fused to HRP was expressed with retained HRP activity, and proved to be functional as a secondary antibody, demonstrating the system’s versatility in producing NBs and conjugated NBs with posttraslational modification that may be required for diverse biotechnological applications.