Expression,purification,and activity analysis of human mitochondrial DNA polymerase gamma
Objective To express and purify human mitochondrial DNA polymerase gama(Polγ),and to analyze its activity for the detection and screening of toxic side effects of nucleoside(acid)antiviral drugs.Methods The human mitochondrial DNA polymerase gamma gene(POLG)was amplified by PCR using the plasmid PCMV-SPORT6-POLG as a template,and cloned into the pPIC9K vector to construct the efficient expression vector pPIC9K-POLG.After enzymatic linearization of the recombinant expression vector,GS115 Pichia pastoris cells were transformed by electroporation transformation method and induced to express at 30℃with 1%methanol.After purification,the expressed protein was identified by SDS-PAGE and Western Blot,and its activity was analyzed using the incorporation method to compare with commercial Pol γ enzyme.Results The size of the POLG gene amplified by PCR was approximately 3.7kb,and the result of the sequencing analysis was consistent with the sequence published in Gen Bank.The expressed Pol γ recombinant protein was a soluble protein with a molecular weight of approximately 138 KD.The recombinant Pol γ enzyme had slightly lower activity than commercial enzymes.Conclusion The recombinant efficient expression vector pPIC9K POLG is constructed,and the recombinant Pol γ enzyme is successfully expressed in yeast cells,establishing an in vitro eukaryotic expression system for Pol γ enzyme.The specific activity of the recombinant enzyme is about 8 U·µL-1,which can be used in the screening and detection of nucleoside(acid)antiviral drugs.
human mitochondrial DNA polymerase gammaGS115 Pichia pastorisexpression and purificationactivity analysis
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