title: [Expression, purification, and bio-activity analysis of fusion protein HBx-EGFP-TLM].
abstract: Hepatitis B virus X protein (HBx) has various functions and plays a crucial role in the development of hepatocellular carcinoma (HCC). However, due to different transfection efficiency levels and experimental approaches, it is difficult to correlate the exact functions of HBx to HBV-associated HCC. In this study, we constructed two prokaryotic expression vectors, pGEX-HBx-EGFP-TLM and pGEX-EGFP-TLM, which expressed HBx-EGFP-TLM and EGFP-TLM fusion proteins respectively. Both vectors contained a coding sequence of TLM transduction motif derived from the PreS2-domain of Hepatitis B Virus surface antigens. In addition, EGFP was expressed as a reporter reflecting the transduction efficiency of TLM. The fusion protein HBx-EGFP-TLM or EGFP-TLM purified from Escherichia coli BL21(DE3) by AKTA Purifier system was incubated with AML12 and SMMC-7721 cells. Both Western blotting and laser confocal results indicated that the translocation motif TLM could lead HBx-EGFP and EGFP into the cytoplasm. Dual-Luciferase Reporter Assay revealed that the activity of mEZH2 promoter could be up-regulated by the recombinant HBx. In conclusion, we expressed a cell-permeable HBx, which could provide a new method to study the functions of HBx.
authors: College of Life Sciences, Northwest A&F University, Yangling 712100, China.
authors: Xiaoyan
authors: X
authors: Shi
authors: Yingying
authors: Y
authors: Zhang
authors: Xiaowei
authors: X
authors: Zhou
authors: Jiansheng
authors: J
authors: Lu
authors: Zekun
authors: Z
authors: Guo
authors: Peitang
authors: P
authors: Huang
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D057846:Cell-Penetrating Peptides / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D006514:Hepatitis B Surface Antigens / Q000235:genetics*; D011498:Protein Precursors / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D015534:Trans-Activators / Q000096:biosynthesis* / Q000235:genetics; D054334:Viral Regulatory and Accessory Proteins
chemical_list: D057846:Cell-Penetrating Peptides; D006514:Hepatitis B Surface Antigens; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C485184:enhanced green fluorescent protein; C057628:hepatitis B virus X protein; C050807:presurface protein 2, hepatitis B surface antigen; D049452:Green Fluorescent Proteins