Effect of post-solubilization conditions on the yield and efficiency of recombinant streptokinase purification at large-scale

Solubilization of inclusion body proteins using low and very low concentrations of chemicals: implications of novel combined chemical treatment designs in enhancement of post‐solubilization target protein purity and biological activity

“…S1 and Table . Having been used as conventional route for IBs' solubilization at large scale, urea at high concentration (4 mol L ‐1 ) solubilized around 76% of the recombinant streptokinase content of the IBs with 72% purity, while the solubilization capacity of medium concentration of urea (2 mol L ‐1 ) and the resulting rSK purity were about 60 and 96%, respectively. Based on these results, solubilization efficiency of guanidinium chloride was less than that of urea at similar concentrations.…”

“…S3), and there was a purity determination discrepancy up to 2% between the two techniques. Following solubilization of IB proteins by traditional chemical methods, chromatographic techniques are normally required for purification of the solubilized target protein in order to reach the required protein purity . Some investigations of the extraction, purification and refolding of recombinant streptokinase from E. coli IBs have been carried out previously.…”

“…Expression of recombinant streptokinase in the form of IBs in E. coli and the stages including bacterial cells disruption, IBs' separation and washings were carried out as described previously . Briefly, recombinant streptokinase was produced as IBs through a batch fermentation process using E. coli W3110 (ATCC 27325) transformed with a plasmid containing SKC‐2 gene (accession number S46536.1) under the control of the tryptophan expression system.…”

“…Escherichia coli has been the host of choice for manufacturing of numerous recombinant proteins, and overexpression of the target proteins in E. coli leads to the accumulation of partially folded protein aggregates, called inclusion bodies (IBs), which contain native‐like secondary structures and classically lack biological activity . Subsequent to bacterial cell rupture or disruption, IBs isolation from the whole cell lysate and reduction of impurities by washing with buffers containing low concentrations of detergents, IB proteins must be solubilized and refolded into an active conformation . However, solubilization of inclusion bodies and refolding of the solubilized proteins into bioactive form is a challenging process since the contained protein molecules are in an aggregate state.…”

“…Proteins expressed as inclusion bodies are typically solubilized in high concentrations of chaotropic agents, such as urea and guanidinium chloride and this process results in denaturation of the native‐like secondary structures in the proteins. Complete unfolding of protein molecules in IBs throughout solubilization enhances the opportunity of aggregation during refolding and consequently leads to low recovery of bioactive protein .…”