Development of a soluble PTD-HPV18E7 fusion protein and its functional characterization in eukaryotic cells.
Though accumulated evidence has demonstrated the transformation capacity of human papillomavirus (HPV) type 18 protein E7, the underlying mechanism is still arguable. Developing a protein transduction domain (PTD)-linked E7 molecule is a suitable strategy for assessing the biological functions of the protein. In the present study, HPV18 E7 protein fused to an N-terminal PTD was expressed in the form of glutathione S-transferase fusion protein in Escherichia coli with pGEX-4T- 3 vector. After glutathione-Sepharose 4B bead affinity purification, immunoblot identification and thrombin cleavage, the PTD-18E7 protein showed structural and functional activity in that it potently transduced the cells and localized into their nuclei. The PTD-18E7 protein transduced the NIH3T3 cells in 30 min and remained stable for at least 24 h. In addition, the PTD-18E7 protein interacted with retinoblastoma protein (pRB) and caused pRB degradation in the transduced NIH3T3 cells. In contrast to the pRB level, p27 protein level was elevated in the transduced NIH3T3 cells. The PTD-18E7 protein gives us a new tool to study the biological functions of the HPV E7 protein.
Yan
Xiaofei
X
Center for Cancer Research, the First Affiliated Hospital, Xi'an Jiaotong University, Xi'an 710061, China.
Walayat
Shah
S
Shi
Qinfeng
Q
Zheng
Jin
J
Wang
Yili
Y
D000818:Animals; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D009856:Oncogene Proteins, Viral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D033903:Periplasmic Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism*; D016160:Retinoblastoma Protein / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012995:Solubility
D004268:DNA-Binding Proteins; C052633:E7 protein, Human papillomavirus type 18; D006360:Heat-Shock Proteins; D009856:Oncogene Proteins, Viral; D033903:Periplasmic Proteins; D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein; C061682:DegP protease; D012697:Serine Endopeptidases