State-of-the-art and novel approaches to mild solubilization of inclusion bodies

Throughout the twenty-first century, the view on inclusion bodies (IBs) has shifted from undesired by-products towards a targeted production strategy for recombinant proteins. Inclusion bodies can easily be separated from the crude extract after cell lysis and contain the product in high purity. However, additional solubilization and refolding steps are required in the processing of IBs to recover the native protein. These unit operations remain a highly empirical field of research in which processes are developed on a case-by-case basis using elaborate screening strategies. It has been shown that a reduction in denaturant concentration during protein solubilization can increase the subsequent refolding yield due to the preservation of correctly folded protein structures. Therefore, many novel solubilization techniques have been developed in the pursuit of mild solubilization conditions that avoid total protein denaturation. In this respect, ionic liquids have been investigated as promising agents, being able to solubilize amyloid-like aggregates and stabilize correctly folded protein structures at the same time. This review briefly summarizes the state-of-the-art of mild solubilization of IBs and highlights some challenges that prevent these novel techniques from being yet adopted in industry. We suggest mechanistic models based on the thermodynamics of protein unfolding with the aid of molecular dynamics simulations as a possible approach to solve these challenges in the future.

Automated summary

Throughout the 21st century, the understanding of inclusion bodies (IBs) has shifted to considering them as a targeted production strategy for recombinant proteins, rather than unwanted by-products. Although IBs can be easily separated and provide a highly pure product, additional steps for solubilization and refolding are necessary. Developing solubilization techniques that maintain correctly folded protein structures without denaturation is a current challenge. Ionic liquids have emerged as promising agents for solubilization, but their adoption in industry is hindered by various challenges. Mechanistic models based on protein unfolding thermodynamics and molecular dynamics simulations are suggested as a potential solution.