Solubilization at high pH results in improved recovery of proteins from inclusion bodies of E. coli

Abstract

BACKGROUND: Solubilization of inclusion bodies using high concentration of chaotropes followed by suboptimal refolding results in lower recovery of bioactive proteins from E. coli. Growing evidence indicates that aggregation of expressed proteins into inclusion bodies is molecular specific in nature and proteins in inclusion bodies have native-like secondary structure. Protecting these pre-existing native-like secondary structures of proteins using mild solubilization conditions is one of the key steps in improved recovery of bioactive protein from inclusion bodies of E. coli.

Potent Malaria Transmission-Blocking Antibody Responses Elicited by Plasmodium falciparum Pfs25 Expressed in Escherichia coli after Successful Protein Refolding

“…High concentrations of chaotropes used for solubilization of inclusion bodies often result in suboptimal protein refolding and reduced recovery of bioactive protein molecules (29,37). First, we opted to employ alternate ways to solubilize inclusion bodies using brief exposure to high pH (29), and the second consideration was protein refolding to obtain Pfs25 in the appropriate conformation (11,22,38).…”

“…First, we opted to employ alternate ways to solubilize inclusion bodies using brief exposure to high pH (29), and the second consideration was protein refolding to obtain Pfs25 in the appropriate conformation (11,22,38). It is well known that the correct pairing of cysteine residues in the protein has important consequences for protein folding, protein stability, and biological function.…”

“…Pfs25 contains 22 conserved cysteine residues, and all 11 disulfide bonds are important for structural integrity of the molecule (11,22). Mispairing of cysteine residues is accompanied by misfolding or aggregation of proteins, requiring solubilization and protein refolding and resulting in low yields of functional molecules (28,29). A previous study on attempts to express Pfs25 in E. coli reaffirmed all the points described above (30).…”

Improving the mda-7/IL-24 refolding and purification process using optimized culture conditions based on the structure characteristics of inclusion bodies

“…The IB solubility will be affected by their structures, which will result in difference in turbidity when IBs were dissolved in denaturation buffer. The turbidity of IB resuspension in the solubilization buffer was chosen as the index to reflect the solubility of IBs, and lower turbidities were associated with higher IB solubilities [25,26]. The turbidity of the suspension was measured at 480 nm.…”

Caracterização da distribuição subcelular e tecidual da proteína KIAA0090 e estudos de seu envolvimento em câncer e resposta a estresses

“…A solubilização desses agregados de proteína, formados em sua maioria por interações hidrofóbicas não nativas entre os intermediários enovelados, com altas concentrações de agentes caotrópicos, por sua vez, geram estruturas espirais aleatórias onde seus aminoácidos hidrofóbicos são expostos, aumentando a propensão de agregação durante o renovelamento (Singh et al, 2008). Vimos que a KIAA0090 apresenta pI de 4,2 e predominância de leucina em sua composição (12,3%), um aminoácido altamente hidrofóbico, sugerindo que estas características físico-químicas, sejam responsáveis pelo fato da proteína se precipitar durante seu renovelamento.…”