Purification of GST-TRAF6 Fusion Proteins from Inculsion Body Expressed in E. coli

Abstract

Abstract:

There was only a minor fraction of GST-TRAF6 fusion proteins were observed soluble when expressed in E.coli. Most of the GST-TRAF6 fusion proteins were present in the inclusion body, in which the expressed proteins aggregate and exist in inactive. To obtain GST-TRAF6 fusion protein in a high yield and high purity, a method to purify the GST-TRAF6 fusion proteins from inclusion body without any loss of its bioactivity was constructed. Firstly, the inclusion body was denatured with 8 mol/L urea. Secondly, the GST-TRAF6 fusion protein in inclusion body were gradually solubilized by gradient dilution renaturation. Once the GST-TRAF6 fusion protein was fully dissolved in renaturation solution, it was conventional to purify the GST-TRAF6 fusion protein with glutathione sepharose 4B. The specificity and the bioactivity of the purified GST-TRAF6 fusion protein were testified by western blot and in vitro ubiquitination assay, respectively. The results indicated that GST-TRAF6 fusion protein were successfully solubilized by gradient dilution renaturation following the denaturation of 8 mol/L urea, the concentration of the purified GST-TRAF6 fusion protein was achieved a concentration of 396 ng/μL with a purity over 90%. The specificity of this GST-TRAF6 fusion proteins were identified with anti-GST western blot. In vitro ubiquitination assay indicated that the GST-TRAF6 fusion protein in a concentration of 17 ng/μL could rapidly catalyze the formation of free ubiquitin chains within 5 minutes. In conclusion, it was found that the GST-TRAF6 fusion protein could be successfully renatured with the method of gradient dilution renaturation with the recovery of its capability of ubiquitin ligase. This results would supply a useful method for the large-scale purification of proteins from inclusion body when expressed in E. coli.

Key words: TRAF6, Protein purification, Inclusion body renaturation, Recombinant protein

Zhang Xixuan, Li Ye, Zhang Zhenqi, Dong Shirui, Zhao Pei, Ruan Haihua. Purification of GST-TRAF6 Fusion Proteins from Inculsion Body Expressed in E. coli[J]. Biotechnology Bulletin, 2014, 0(8): 182-188.

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