Construction,Expression and purification of human telomerase reverse transcriptase in E.coli

Objective To construct,express,and purify human telomerase reverse transcriptase(hTERT) functional motifs protein.Methods A 1.3kb foreign DNA insert of telomerase catalytic subunit gene containing all 8 motifs was cloned and amplified by PCR into expression vector of pET-32a.The recombinant plasmid was induced by IPTG for 4 h and produced 59 kD recombinant protein which appeared in the form of inclusion body.This inclusion body was dissolved in 8 mol/L urea and purified by affinity chromatography using Ni-NTA resin under denaturing conditions.Purified hTERT protein was identified by SDS-PAGE and Western-Blot analysis.Results The obtained target gene fragment was 1 310bp.The recombinant pET32a-hTERT was expressed in E.coli at high level as inclusion body,accounting for 18% of the total bacterial proteins.The relative molecular mass of the protein appeared to be 59 kD by SDS-PAGE.Purification of recombinant protein was carried out by Ni-NTA affinity chromatography.The identification of recombinant hTERT was confirmed by SDS-PAGE and Western-Blot analysis.Conclusion Identification results revealed that expression plasmid pET32a-hTERT was constructed successfully and recombinant protein of hTERT functional region was expressed and purified correctly.