MOLECULAR CLONING OF hTRT CATALYTIC DOMAIN FROM HeLa CELLS AND ITS EXPRESSION IN E Coli AND PURIFICATION
中国医学科学杂志(英文版)2000年0卷第3期 页码:170-174
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MOLECULAR CLONING OF hTRT CATALYTIC DOMAIN FROM HeLa CELLS AND ITS EXPRESSION IN E Coli AND PURIFICATION
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中图分类号:R392
纸质出版日期:2000
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MOLECULAR CLONING OF hTRT CATALYTIC DOMAIN FROM HeLa CELLS AND ITS EXPRESSION IN E Coli AND PURIFICATION[J]. 中国医学科学杂志(英文版), 2000,0(3):170-174.
MOLECULAR CLONING OF hTRT CATALYTIC DOMAIN FROM HeLa CELLS AND ITS EXPRESSION IN E Coli AND PURIFICATION[J]. Chinese medical sciences journal, 2000, 0(3): 170-174.
MOLECULAR CLONING OF hTRT CATALYTIC DOMAIN FROM HeLa CELLS AND ITS EXPRESSION IN E Coli AND PURIFICATION[J]. 中国医学科学杂志(英文版), 2000,0(3):170-174.DOI:
MOLECULAR CLONING OF hTRT CATALYTIC DOMAIN FROM HeLa CELLS AND ITS EXPRESSION IN E Coli AND PURIFICATION[J]. Chinese medical sciences journal, 2000, 0(3): 170-174.DOI:
MOLECULAR CLONING OF hTRT CATALYTIC DOMAIN FROM HeLa CELLS AND ITS EXPRESSION IN E Coli AND PURIFICATION
摘要
Abstract
To investigate the expression of telomerase gene hTRT mRNA in HeLa cells and to obtain hTRT protein for futher 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 proteins were purified by Ni NTA chromatography and stained by western blotting Results. An approximately 620bp fragment was generated and cloned into pBluescript SK+between SalI 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×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×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 staining 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
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RADIATION- INDUCED PROGRESSIVE DECREASING IN THE EXPRESSION OF REVERSE TRANSCRIPTASE GENE OF hEST2 AND TELOMERASE ACTIVITY
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