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.
Subject(s)
Amino Acid Motifs , Genetics , Cell-Penetrating Peptides , Genetics , Escherichia coli , Genetics , Metabolism , Genetic Vectors , Genetics , Green Fluorescent Proteins , Genetics , Hepatitis B Surface Antigens , Genetics , Protein Precursors , Genetics , Recombinant Fusion Proteins , Genetics , Trans-Activators , GeneticsABSTRACT
AIM:To construct eukaryotic expression plasmids encoding Sox2 and Sox2 K247R and identify their expression and SUMOylation. METHODS: With gift plas-mid encoding Sox2 gene as a template, Sox2 K247R was obtained by overlapping extension PCR, followed by construction of pCMV-HA-Sox2 and pCMV-HA-Sox2 K247R. After enzyme digestion analysis and DNA sequencing, these two constructs were transfected or co-transfected with pC-MV-Myc-SUMO1 into 293 FT cells by lipofectin method. Western blot was employed to analyze expression and SUMO ylation of Sox2. RESULTS: It was revealed that eukaryotic expression vectors were constructed with correct sequence, where in mutant Sox2, the AAG codon was switched to CGG codon. Western blot results showed that good expression of both wt and mut Sox2, of which the latter could not be modified by SUMO1. CONCLUSION: Successful construction and expression of Sox2 and Sox2 K247R. Sox2 could be SUMO lyated in vitro but Sox2 K247R not.