Preparation of a novel telomerase inhibitory protein LPTS-L / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 852-857, 2007.
Article
in Chinese
| WPRIM
| ID: wpr-327935
ABSTRACT
The gene for LPTS is originally cloned as a human liver-related putative tumor suppressor (LPTS) gene that encodes a full length protein of 328 amino acids (LPTS-L). LPTS-L is also identified as a telomerase inhibitor to regulate telomere length in the cells. To facilitate the functional and structural studies of LPTS-L protein, the cDNA for LPTS-L was cloned into the expression vector pET-24 in frame to generate a recombinant plasmid pET-24-LPTS. The LPTS-L protein was expressed in E. coli BL21 solublely, and purified by Ni Sepharose affinity chromatography which, however, is not fit for large scale protein purification. The gene of LITS-L was then PCR amplified to remove the 6 x His tag, and cloned into pET-24a. The non-fusion protein of LPTS-L was expressed in E. coli B21, and purified by phosphocellulose P11 chromatography. The purity of LPTS-L protein was about 55% after that procedure,and arrived at 80% after second purification by Sephadex G-100 chromatography. Western Blotting analysis showed that the band reflects the specific binding of anti-LPTS antiserum against the purified LPTS-L protein. The TRAP assay was performed to detect the telomerase inhibitory activity of LPTS-L protein in vitro. It was observed that the purified LPTS-L inhibited the activity of telomerase greatly, similarly with that of LPTS-L protein purified by Ni Sepharose 4B. Our results suggest that phosphocellulose P11 plus Sephadex G-100 chromatography could substitute for Ni Sepharose 4B affinity chromatography for preparation of purified LPTS-L protein. Through this study, a technique for preparation of LPTS-L protein in a large scale is established.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Recombination, Genetic
/
Recombinant Proteins
/
Cloning, Molecular
/
Telomerase
/
Tumor Suppressor Proteins
/
Escherichia coli
/
Genetics
/
Metabolism
Limits:
Animals
/
Humans
Language:
Chinese
Journal:
Chinese Journal of Biotechnology
Year:
2007
Type:
Article
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