Chemical Research in Chinese Universities ›› 2009, Vol. 25 ›› Issue (5): 690-694.

• Articles • Previous Articles     Next Articles

Refolding and Purification of Yeast Acetyl-CoA Carboxylases CT Domain Expressed as Inclusion Bodies in Escherichia coli

YANG Xue-ying, TAO Jin, ZHENG Liang-yu*, WANG Rui-jian, ZHAO Ke and CAO Shu-gui*   

  1. Key Laboratory for Molecular Enzymology and Engineering, Ministry of Education, Jilin University, Changchun 130021, P. R. China
  • Received:2008-08-08 Revised:2008-10-16 Online:2009-09-25 Published:2009-12-07
  • Contact: ZHENG Liang-yu, E-mail: lyzheng@jlu.edu.cn; CAO Shu-gui, E-mail: caosg@jlu.edu.cn
  • Supported by:

    Supported by the National Natural Science Foundation of China(Nos.20432010, 20672045 and 30870539).

Abstract:

Acetyl-CoA carboxylase(ACCase) is a crucial enzyme in fatty acid synthesis, by regulating the first committed step in the process. Therefore, it is a potential target for the development of new compounds against   obesity or as herbicides. The cDNA encoding yeast ACCase CT domains(YCTs) from Saccharomyces cerevisiae was amplified by RT-PCR and inserted into the vector PET28a(+) for bacterial expression of YCT fused to N-terminal His-tag(YCT-his6). YCTs-his6 was expressed in Escherichia coli BL21(DE3) PLys as inclusion bodies, which was solubilized in 8 mol/L urea. Ni-agarose chromatography was used to purify the inclusion bodies under denaturing condition. Correct refolding was achieved via systematic dialysis to remove the denaturant gently in the presence of 0.5 mmol/L Triton X-100. The low concentration Triton X-100 was included in the refolding buffer to increase the solubilization and enhance dimeric formation of refolding proteins. The activity of the refolded YCT-his6 was 1.2 U/mg as measured in a spectrophotometric assay using malonyl-CoA as the substrate. To our knowledge, it is the first time that the bioactive YCT-his6 has been expressed successfully in E. coli and isolated from their inclusion bodies.

Key words: Carboxyltransferase domain; Expression; Inclusion body; Refolding