Comparative study to develop a single method for retrieving wide class of recombinant proteins from classical inclusion bodies
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The formation of inclusion bodies (IBs) is considered as an Achilles heel of heterologous protein expression in bacterial hosts. Wide array of techniques has been developed to recover biochemically challenging proteins from IBs. However, acquiring the active state even from the same protein family was found to be an independent of single established method. Here, we present a new strategy for the recovery of wide sub-classes of recombinant protein from harsh IBs. We found that numerous methods and their combinations for reducing IB formation and producing soluble proteins were not effective, if the inclusion bodies were harsh in nature. On the other hand, different practices with mild solubilization buffers were able to solubilize IBs completely, yet the recovery of active protein requires large screening of refolding buffers. With the integration of previously reported mild solubilization techniques, we proposed an improved method, which comprised low sarkosyl concentration, ranging from 0.05 to 0.1% coupled with slow freezing (− 1 °C/min) and fast thaw (room temperature), resulting in greater solubility and the integrity of solubilized protein. Dilution method was employed with single buffer to restore activity for every sub-class of recombinant protein. Results showed that the recovered protein’s activity was significantly higher compared with traditional solubilization/refolding approach. Solubilization of IBs by the described method was proved milder in nature, which restored native-like conformation of proteins within IBs.
KeywordsProkaryotic expression system Harsh inclusion bodies Mild solubilization Sarkosyl (SLS) Freeze/thaw Refolding
Authors would like to thank Dr. Muhammad Kamran Raja (Sun Yat-Sen Cancer Center (SYSCC), Sun Yat-Sen University) for providing excellent technical assistance. This study was supported by grants from The National Natural Science Foundation of China (81301995; 81472836).
Compliance with ethical standards
All authors have seen the content of this manuscript, and have no conflict of interest to disclose.
This article does not contain any studies with human participants or animals performed by any of the authors.
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