2015年2月22日，我院熊思东教授在Microbial Cell Factories杂志发表了题为“A single freeze-thawing cycle for highly efficient solubilization of inclusion body proteins and its refolding into bioactive form” 的研究论文，该论文发现了一种高效简易的溶解包涵体并获得生物活性蛋白的新方法。
齐兴梅讲师为该论文的第一作者。参与该论文研究的还有研究生孙毅凡等。熊思东教授为该论文的通讯作者。该项研究由国家教育部博士点基金资助项目(20133201120019)，国家自然科学基金(31400789)，江苏省创新团队等基金资助。
文章摘要：
Background: 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.
Results: Here 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.
Conclusions: Mild 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.