Simultaneous refolding and purification of MO-CBP2 IBs using urea gradient

Nowadays heterologous expression of proteins plays a key role in biotechnology. Inclusion body formation in heterologous expression of proteins, especially expression of eukaryotic proteins in prokaryotic hosts including E. coli, is one of the most laborious challenges for researchers. High expression of protein, its specific conformation, disulfide bonds and protein charge are account for inclusion body formation. It has been reported that some inclusion bodies have biological activity but in most cases they should be renatured and soluble. Refolding processes are often not only multi-steps and time-consuming but also have low yields. In this study for the first time MO-CBP2 from Moringa Oleifera seed, successfully expressed heterologously in the E. coli but formation of inclusion bodies was a great challenge. Different methods were carried out for refolding the protein, more efficient one was Nickel sepharose column affinity chromatography with urea gradient 8-0 M that result in simultaneous refolding and purification of the protein in fewer steps. This method yields a considerable amount of pure and renatured protein and it is recommended as a convenient and efficient method.