Isolation, Purification And Identification Of The Human DNA Polymerase γ

AIM: To purify and identify the mitochondrial DNA polymeraseγ(polymeraseγ,Polγ) from Hela cell.MATERIALS AND METHODS: For screening anti-HBV compounds without undesirable side effects, ion exchange chromatography on DEAE, P11 and HTP was used to separate Polγfrom the fraction of Hela cell. The concentration of the protein was measured through Bradford method. SDS-PAGE was performed to determine the molecular weight of the subunit of Polγ. With the method ofα-32P-dTTP incorporating, the activity of Polγwas counted by liquid scintillation spectrometer.RESULTS: Polγwas purified by 150-fold to apparent homogeneity with 6% yield. SDS-PAGE indicated the presence of one subunit of 140KD and Western-Blot blotting identified the specificity. Total activity and specific activity of Polγwere 4.81ukat/mg and 36.17 ukat/mg respectively by a series of chromatography.CONCLUSION: Polγcan be purified through ion exchange chromatography, which was activated and can be used as a target to detect the toxicity of some compounds to mitochondria in vitro.