Molecular cloning of hTRT catalytic domain from HeLa cells and its expression in E. coli and purification.
OBJECTIVE
To investigate the expression of telomerase gene hTRT mRNA in HeLa cells and to obtain hTRT protein for further study.


METHODS
The gene for encoding hTRT catalytic domain was cloned based on RT-PCR amplification from HeLa cells and sequenced. The cloned hTRTcDNA was in-frame inserted into His-tag fusion expression vector pEK318. The His-tag hTRT fusion protein were purified by Ni-NTA chromatography and stained by western blotting.


RESULTS
An approximately 620bp fragment was generated and cloned into pBluescript SK + between SaII and BamHI sites. DNA sequencing showed the isolated fragment was consistent to those reported. SDS-PAGE present that a 17kDa protein was expressed stably in E. coli JM109 harboring pEKTRT344 containing 6 x His-tag and hTRT 150aa, and the expression level of the protein was about 26% of the total bacterial proteins, while the expression of pEKTRT containing 6 x His-tag and hTRT 243aa was only detectable as 27 kDa band in western blotting. Both of fusion proteins were purified by Ni-NTA chromatography and showed single band( > 95% purifity) in Coomassie Brilliant staininng. Western-blotting confirmed that two proteins could be recognized by the Ni-NTA AP conjugate.


CONCLUSIONS
The hTRT catalytic domain was highly conserved. The expressed hTRT protein contained recognizable His-tag, telomerase-specific and strong antigenic epitops, which may be convenient for further investigation.
Ying
J
J
Molecular Oncology Laboratory, Department of Pathology, Beijing Medical University, Beijing 100083.
Zhang
B
B
Hou
L
L
Wu
B
B
D020134:Catalytic Domain / Q000235:genetics*; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D006367:HeLa Cells; D006801:Humans; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D019098:Telomerase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D019098:Telomerase