title: A single freeze-thawing cycle for highly efficient solubilization of inclusion body proteins and its refolding into bioactive form.
abstract: 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.
authors: Jiangsu Key Laboratory of Infection and Immunity, Institutes of Biology and Medical Sciences, Soochow University, Suzhou, Jiangsu, 215123, China. xmqi@suda.edu.cn.
authors: Xingmei
authors: X
authors: Qi
authors: Jiangsu Key Laboratory of Infection and Immunity, Institutes of Biology and Medical Sciences, Soochow University, Suzhou, Jiangsu, 215123, China. sunyf6796@126.com.
authors: Yifan
authors: Y
authors: Sun
authors: Jiangsu Key Laboratory of Infection and Immunity, Institutes of Biology and Medical Sciences, Soochow University, Suzhou, Jiangsu, 215123, China. sdxiongfd@126.com.
authors: Sidong
authors: S
authors: Xiong
mesh_terms: D020134:Catalytic Domain; D004926:Escherichia coli / Q000378:metabolism; D005615:Freezing; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000378:metabolism*; D053508:Matrix Metalloproteinase 12 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D012995:Solubility; D013696:Temperature; D014508:Urea / Q000737:chemistry
chemical_list: D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D014508:Urea; D053508:Matrix Metalloproteinase 12