Preparation of a novel telomerase inhibitory protein LPTS-L.
- Author:
Chu WU
1
;
Liang DA
;
Guang-Ming CHEN
;
Fang ZHANG
;
Mu-Jun ZHAO
Author Information
1. Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Cloning, Molecular;
Escherichia coli;
genetics;
metabolism;
Humans;
Recombinant Proteins;
biosynthesis;
genetics;
isolation & purification;
Recombination, Genetic;
Telomerase;
antagonists & inhibitors;
Tumor Suppressor Proteins;
biosynthesis;
genetics
- From:
Chinese Journal of Biotechnology
2007;23(5):852-857
- CountryChina
- Language:Chinese
-
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.
