Transcriptional activation function of a new protein-XTP11 in yeast cells / 解放军医学杂志

ABSTRACT

Objective To construct the yeast expression vector of a new gene transactivated by hepatitis B virus X protein (XTP11), and to explore the feasibility of cloning the hepatocyte proteins interacting with XTP11 protein by yeast two hybrid system. Methods The XTP11 gene was amplified by polymerase chain reaction (PCR) with specific primers, and the amplified fragment was subcloned into the Nco I/BamH I sites (5′ ends) of yeast expression vector pGBKT7,which was named as pGBKT7(-)-XTP11 encoding fusion proteins of full length of XTP11 and DNA binding domain of yeast protein GAL4. The pGBKT7 (-)-XTP11 plasmid was transformed in the yeast cells AH109 and the expression of XTP11 protein in yeast cells was detected by Western blotting assay. Then the yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp and SD/-Trp-His-Ade) containing X-?-gal, and their autonomous activation was tested. Results The yeast expression vector of XTP11 gene was constructed and the fusion protein in yeast cells was examined. The yeast cells transformed with pGBKT7-XTP11 plasmid could grew well on both of the media, and turned blue on medium containing X-?-gal for the production X-?-galactosidase. This phenomenon suggested that XTP11 protein acted to substitute the functional yeast protein GAL4 activation domain (AD) to activate transcription of reporter genes (ADE2, HIS3, MEL1 and LacZ). Conclusion The XTP11 protein fused to the GAL4 DNA-binding domain functioned as a transcriptional activation domain in yeast, and the transcriptional activation function of XTP11 protein in yeast might restrain researchers to use yeast two hybrid system to clone hepatocyte proteins interacting with XTP11 protein.