[Preparation of a novel telomerase inhibitory protein LPTS-L].
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.
Wu
Chu
C
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Da
Liang
L
Chen
Guang-Ming
GM
Zhang
Fang
F
Zhao
Mu-Jun
MJ
D000818:Animals; D018797:Cell Cycle Proteins; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011995:Recombination, Genetic; D019098:Telomerase / Q000037:antagonists & inhibitors*; D025521:Tumor Suppressor Proteins / Q000096:biosynthesis* / Q000235:genetics
D018797:Cell Cycle Proteins; C440808:PINX1 protein, human; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; D019098:Telomerase