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  • Title: [Expression of a human myofibrillogenesis regulator 1 gene in E. coli and its immunogenicity].
    Author: Li TB, Hu Y, Feng S, Zuo ZY, Wang YG.
    Journal: Zhongguo Yi Xue Ke Xue Yuan Xue Bao; 2005 Feb; 27(1):42-7. PubMed ID: 15782492.
    Abstract:
    OBJECTIVE: To study the expression of human myofibrillogenesis regulator 1 (MR-1) gene in E. coli and obtain the MR-1 protein and its antibody for further investigation of its biological function. METHODS: Expression vectors pGEX-5X-1, pET30a (+), and pET24a (+), as well as host strain E. coli BL21 (DE3) and BL21-CodonPlus (DE3) -RIL were used for expression of MR-1. MR-1 N-terminal with GST or T7-tag or C-terminal with His-tag, separately, or N terminal with T7-tag and C terminal with His-tag, simultaneously, were fused in plasmids pGEX-5X-1, pET30a (+) , and pET24a (+). The expressed MR-1-T protein, separated and purified by preparative SDS-PAGE, was applied to immunize the rabbits. The titer of the antibody was assayed by ELISA and its immunogenicity was tested by Western blot with pcDNA3/MR-1 transfected human breast cancer cell MCF7. RESULTS: The MR-1 protein was successfully expressed as inclusion body by fusing its N-terminal with T7-tag in E. coli BL21-CodonPlus (DE3) -RIL. MR-1 protein was purified by electro-elution from SDS-PAGE gel. Using this purified protein, polyclonal antibody in rabbit against MR-1 was essentially generated. ELISA and Western blot showed the titer of this antibody was about 1:10(5) with high immunogenicity. CONCLUSIONS: The N-terminal fusion tag is the most important mechanism for MR-1 expression. The polyclonal antibody of the generated MR-1 protein in E. coli may be applied for its further biological function studies.
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