Streamlined on-column refolding and purification of nanobodies from inclusion bodies expressed as fusion proteins

This study introduces an efficient on-column refolding and purification method for preparing nanobodies (Nbs) expressed as inclusion bodies and fusion proteins. The HisTrap (TM) FF system was successfully employed for the purification of the fusion protein FN1-Delta I-CM-2D5. The intein Delta I-CM cleavage activity was activated at 42 degrees C, followed by incubation for 4 h. Leveraging the remarkable thermal stability of Nbs, 2D5 was further purified through heat treatment at 80 degrees C for 1h. This method yielded up to 107.2 mg of pure 2D5 with a purity of 99.2 % from just 1L of bacterial culture grown in a shaker flask. Furthermore, this approach successfully restored native secondary structure and affinity of 2D5. Additionally, the platform was effectively applied to the refolding and purification of a polystyrene-binding nanobody (B2), which exhibited limited expression in the periplasmic and cytoplasmic spaces of E. coli. This endeavor resulted in the isolation of 53.2 mg of pure B2 Nb with a purity exceeding 99.5 % from the same volume of bacterial culture. Significantly, this approach restored the native secondary structure of the Nbs, highlighting its potential for addressing challenges associated with expressing complex Nbs in E. coli. Overall, this innovative platform provides a scientifically rigorous and reproducible method for the efficient preparation of Nbs, offering a valuable tool for antibody research and development.