[Expression of recombinant human doppel protein and analysis of its cytotoxic activities].
Doppel (Dpl) is a newly identified PrP-associated protein. In this study, specific primers for human PRND gene were designed based on the human PRND cDNA sequence encoding human Dpl protein reported in the GenBank. The full-length PRND gene sequence with 531 bp long was obtained from DNA of human peripheral leucocytes as the template by polymerase chain reaction (PCR). After verified by sequence analysis, the PCR product was inserted into a prokaryotic-expressing vector and then transformed into E. coli JM109. The recombinant human Doppel protein (rhDpl) was expressed as inclusion bodies after IPTG induction, with the yield of more than 60% of total bacterial proteins. The rhDpl protein was purified by Ni2+ affinity chromatography and cleaved by hydroxylamine. SDS-PAGE revealed the molecular weight of the purified rhDpl protein was about 15 kD. Trypan blue and MTT assays identified that rhDpl in vitro inhibited the growth of human neuroblastoma cell line SH-SY5Y at concentrations > or =50 microg/mL and human cervical cancer cell line HeLa at concentrations > or =100 microg/mL, showing remarkably dose-and time-dependant manners. Hoechst33342-staining of SH-SY5Y cells treated with rhDpl showed massive apoptosis under fluorescent microscope. These results indicate that Dpl protein possesses cytotoxic activity in vitro, with obvious tissue-specific characteristics. This study provides the foundation for further study of Dpl biological functions in vitro and in vivo.
Li
Ping
P
State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China.
Han
Jun
J
Shan
Bing
B
Lei
Yan-jun
YJ
Zhou
Wei
W
Jiang
Hui-ying
HY
Dong
Xiao-ping
XP
D017209:Apoptosis / Q000187:drug effects; D001562:Benzimidazoles / Q000737:chemistry; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D058851:GPI-Linked Proteins; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D011328:Prions / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology
D001562:Benzimidazoles; D058851:GPI-Linked Proteins; C501125:PRND protein, human; D011328:Prions; D011994:Recombinant Proteins; C017807:bisbenzimide ethoxide trihydrochloride