Aggregation is a serious obstacle to recovery of biologically active proteins from inclusion bodies (IBs) produced by E. coli. High hydrostatic pressure was described as an interesting tool to solubilize and refold aggregated proteins. In order to obtain the refolding of aggregated and insoluble protein, cruzain IBs were subjected to compression (2 kbar) for 16 h and the effects of changes in ratio and concentration of oxido‐shuffling reagents and concentration of guanidine hydrochlorid were assayed. A 50% yield in refolding of the native cruzain was obtained using optimal conditions of the refolding buffer (Tris‐HCl buffer, pH 8.0, 0.75 M Gdn‐HCl, 2 mM EDTA). Additives in the refolding buffer did not further improve cruzain refolding yields. The presence of Bis‐ANS, a hydrophobic extrinsic fluorescent dye, which has been previously described to be able to suppress protein aggregation, surprisingly abolished completely cruzain refolding. The refolded cruzain presents enzymatic activity hydrolyzing a specific substrate (Z‐FR‐MCA). We demonstrated that high pressure can successfully refold cruzain with biological activity from insoluble aggregates in inclusion bodies.
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