Abstract
BackgroundMild solubilization of inclusion bodies has attracted attention in recent days, with an objective to preserve the existing native-like secondary structure of proteins, reduce protein aggregation during refolding and recovering high amount of bioactive proteins from inclusion bodies.ResultsHere we presented an efficient method for mild solubilization of inclusion bodies by using a freeze-thawing process in the presence of low concentration of urea. We used two different proteins to demonstrate the advantage of this method over the traditional urea-denatured method: enhanced green fluorescent protein (EGFP) and the catalytic domain of human macrophage metalloelastase (MMP-12_CAT). Firstly, PBS buffer at pH 8 containing different molar concentration of urea (0-8 M) were used to solubilize EGFP and MMP-12-CAT inclusion bodies and the solubility achieved in 2 M urea in PBS buffer by freeze-thawing method was comparable to that of PBS buffer containing 8 M urea by traditional urea-denatured method. Secondly, different solvents were used to solubilize EGFP and MMP-12_CAT from inclusion bodies and the results indicated that a wide range of buffers containing 2 M urea could efficiently solubilize EGFP and MMP-12_CAT inclusion bodies by freeze-thawing method. Thirdly, the effect of pH and freezing temperature on the solubility of EGFP and MMP-12_CAT inclusion bodies were studied, revealing that solubilization of inclusion bodies by freeze-thawing method is pH dependent and the optimal freezing temperature indicated here is −20°C. Forth, the solubilized EGFP and MMP-12_CAT from inclusion bodies were refolded by rapid dilution and dialysis, respectively. The results showed that the refolded efficiency is much higher (more than twice) from freeze-thawing method than the traditional urea-denatured method. The freeze-thawing method containing 2 M urea also effectively solubilized a number of proteins as inclusion bodies in E.coli.ConclusionsMild solubilization of inclusion body proteins using the freeze-thawing method is simple, highly efficient and generally applicable. The method can be utilized to prepare large quantities of bioactive soluble proteins from inclusion bodies for basic research and industrial purpose.
Highlights
Mild solubilization of inclusion bodies has attracted attention in recent days, with an objective to preserve the existing native-like secondary structure of proteins, reduce protein aggregation during refolding and recovering high amount of bioactive proteins from inclusion bodies
enhanced green fluorescent protein (EGFP) (27 kDa) could be produced in both active form and inactive inclusion bodies in E. coli expression system depending on inducing protein over-expression conditions [28,29]
Previous study have shown that the catalytic domain of human macrophage metalloelastase (MMP-12_CAT) are expressed at high levels in E. coli only as inclusion bodies, and its active form could be obtained by using high concentration of denaturing reagents denaturing and refolding [3,31]
Summary
Mild solubilization of inclusion bodies has attracted attention in recent days, with an objective to preserve the existing native-like secondary structure of proteins, reduce protein aggregation during refolding and recovering high amount of bioactive proteins from inclusion bodies. Mild solubilization of inclusion body aggregates has attracted attention in recent days, it allows for preservation of existing native-like secondary structure of proteins, reduces protein aggregation during refolding and allows for recovery of high amount of bioactive proteins from inclusion bodies [20,21]. Many novel solubilization methods without using high concentration of denaturing reagents have been developed for solubilization and recovery of bioactive protein form inclusion bodies [13,14,22,23,24]
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