Background

Expression and purification of recombinant proteins from bacterial, viral, and eukaryotic expression systems is routinely carried out in the biotechnological industry, especially in the areas of development and commercialization of successful protein-based drug products. In addition, the expression and purification of recombinant proteins to study protein-protein, protein-nucleic acid, and protein-drug interactions, is routinely carried out in both academic institutions and the biotechnological industry. The inherent high aggregation tendency of proteins at various stages of expression, purification, and storage has significant impact upon product quality, safety, and efficacy. Unlike eukaryotes, the reduced environment of the bacterial cytosol and the lack of eukaryotic chaperones and post-translational machineries limit the efficient protein folding capability of a bacterial system. Due to these limitations, the huge expression of recombinant proteins in Escherichia coli (E. coli) often results in aggregation in inclusion bodies (Williams et al., 1982; Freedman and Wetzel, 1992; Chrunyk et al., 1993). The expression of the recombinant proteins at high translational rates exhausts the bacterial protein quality control system, resulting in the aggregation of partially folded and misfolded protein molecules to form inclusion bodies (Carrio and Villaverde, 2005). Formation of inclusion bodies poses a great challenge in the production and purification of recombinant proteins using E. coli as host. Purification of native-like proteins from inclusion bodies is extremely difficult. Despite the intensive processing, including the isolation of inclusion bodies from the cell, solubilization using denaturants, followed by refolding, the finally purified proteins tend to re-aggregate and show minimal activity. Protein aggregation in inclusion bodies can be reduced by decreasing the temperature of growing bacterial culture up to 16°C and reducing the inducer concentration. Although these efforts can be helpful for certain proteins, for most others the aggregation is triggered when bacterial cells are lysed during the purification process.

The detergent gradient approach discussed here for the purification of Crimean Congo Hemorrhagic fever virus (CCHFV) nucleocapsid protein (CCHFVNP) separates the early formed inactive protein aggregates from the correctly folded active protein and prevents further aggregation in the native protein sample. The correctly folded protein is retained in the appropriate microenvironment of the gradient, making the protein resistant to aggregation, despite numerous freeze–thaw cycles. The approach can be customized for the purification of any recombinant protein that is prone to aggregation. This native protein purification method produces a significant amount of pure protein without denaturation and refolding steps, commonly used in most protein purification procedures. The yield of the active protein produced by this method depends upon the aggregation tendency of the protein, which varies from protein to protein. If a protein has more aggregation tendency, the aggregates will move towards the bottom of the gradient, and little active protein stays on the top. For our protein (CCHFNP), almost 60% was aggregated, and the remaining 40% was active, which was pooled together and stored for later use. The approach will be of potential significance to the biotechnological industry, which faces challenges of protein aggregation at different stages of development and commercialization of protein-based drug products (Wang and Roberts, 2018).