DNA聚合酶γ的提取、纯化和鉴定
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张颖慧, 林菊生, 李 岩, 高琳琳, 王晓燕
人宫颈癌细胞;DNA聚合酶γ;线粒体;离子交换层析,张颖慧,林菊生,李岩,高琳琳,王晓燕,张颖慧,通讯作者:,Isolation,purificationandidentificationofDNApolymerasegamma,Ying-HuiZhang
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张颖慧, 林菊生, 李岩, 高琳琳, 王晓燕, 华中科技大学同济医学院附属同济医院肝病研究所 湖北省武汉市 430030
张颖慧, 2005年华中科技大学同济医学院硕士, 主要从事肝脏病学研究.
国家自然科学基金资助项目, No. 30330680
通讯作者: 林菊生, 430030, 湖北省武汉市, 华中科技大学同济医学院附属同济医院肝病研究所. linjusheng2001@163.com
电话: 027-83662578
收稿日期: 2007-09-17 修回日期: 2007-11-06
Isolation, purification and identification of DNA polymerase gamma
Ying-Hui Zhang, Ju-Sheng Lin, Yan Li, Lin-Lin Gao, Xiao-Yan Wang
Ying-Hui Zhang, Ju-Sheng Lin, Yan Li, Lin-Lin Gao, Xiao-Yan Wang, Institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China
Supported by: National Natural Science Foundation of China, No. 30330680
Correspondence to: Dr. Ju-Sheng Lin, Institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China. linjusheng2001@163.com
Received: 2007-09-17 Revised: 2007-11-06
Abstract
AIM: To purify and identify the mitochondrial DNA polymerase gamma (polymerase γ, Pol γ) from HeLa cells.
METHODS: Ion exchange chromatography was used to isolate Pol γ from HeLa cells. Protein concentration was measured using the Bradford method. SDS-PAGE was performed to determine the molecular weights of the subunits of Pol γ. Following the incorporation of α-32P-dTTP, the activity of Pol γ was counted using a liquid scintillation spectrometer.
RESULTS: Pol γ was purified by 150-fold to apparent homogeneity, with a 6% yield. SDS-PAGE indicated the presence of one subunit of 140 kDa, and Western blotting identified the specificity. Total activity and specific activity of Pol γ were determined to be 4.81 U and 36.17 U/mg, respectively, by chromatography.
CONCLUSION: Pol γ can be purified by ion exchange chromatography. It can then be activated and used as a target to detect the toxicity of some compounds to mitochondria in vitro ......
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