[Cloning and expression of recombinant mitochondrial Delta1-120 apoptosis-inducing factor and its enhancive effect on apoptosis of hepatocellular carcinoma cell line SMMC-7721].
OBJECTIVE
Mitochondria play a key role in cell apoptosis, and apoptosis-inducing factor (AIF) is a kind of apoptotic protein located in mitochondria. The research on mitochondrial protein can be helpful for elucidating the role of mitochondria in apoptosis. This study was to clone and express recombinant human Delta1-120 AIF and validate its biological activities of binding DNA and inducing nuclear apoptosis.


METHODS
A human AIF gene fragment of 1515 bp(mitochondrial localization sequence was deleted) was amplified from SMMC-7721 cells by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into pET32a(+) vector to construct recombinant plasmid pET32a-AIF. The recombinant plasmid was transfected into E.coli BL2l (DE3). AIF expression was induced by isoprophylthio-beta-D-galactoside (IPTG), and detected by SDS-PAGE and Western blot. AIF protein was purified by Ni afinity chromatography and then renatured. The biological activity of renatured AIF protein was detected by electrophoretic mobility shift assay (EMSA) and Hoechst staining.


RESULTS
The 1.5 kb AIF gene was successfully isolated, and cloned into pET32a(+) vector. Plasmid pAIF was identified by restrictive enzyme analysis and sequencing. Recombinant E.coli. strains expressing AIF were obtained. AIF protein amounted to 11% of the total bacterial protein when induced with IPTG at 37 degrees Celsius for 4 h. AIF was specially recognizing by anti-AIF and anti-his antibody. The purity of purified protein reached over 95%. After renaturation, AIF protein binded DNA and induced nuclear apoptosis.


CONCLUSIONS
AIF protein with high purity and biological activity was obtained by the method described above.
Key Laboratory of Laboratory Medical Diagnostics of Ministry of Education, Chongqing University of Medical Science, Chongqing, 400016, PR China.
Yu-Rong
YR
Fu
Zheng-Jun
ZJ
Yi
Yu-Rong
YR
Yan
Zong-Yin
ZY
Qiu
D017209:Apoptosis; D051033:Apoptosis Inducing Factor / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000473:pathology*; D045744:Cell Line, Tumor; D004273:DNA, Neoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000473:pathology*; D010957:Plasmids; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
C494829:AIFM1 protein, human; D051033:Apoptosis Inducing Factor; D004273:DNA, Neoplasm; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins