Fig. 3
From Glycerol Stock to Lab-Scale Production: A Conventional Nb Production Protocol. Various systems are available for nanobody production, including bacterial, yeast, mammalian, and plant expression systems. Among these, the bacterial expression system, particularly using E. coli, is the most common due to its simplicity, cost-effectiveness, and high yield. In this system, LB medium supplemented with glucose, ampicillin, and MgCl2 is inoculated with glycerol stock and incubated overnight. The culture then seeds TB medium for a few hours before nanobody (Nb) expression is induced with IPTG overnight. Post-expression, cells are centrifuged and lysed to extract Nbs. The extract undergoes IMAC purification, followed by imidazole elution, with an optional size exclusion chromatography. Quality is confirmed through SDS-PAGE, Coomassie staining, and western blotting. Adapted illustration [3]