Expression of human alcohol dehydrogenase ADH1B2 in Pichia pastoris and its activity analysis

Human alcohol dehydrongenase gene ADH1B2 was obtained by RT-PCR using total RNA from liver as template. Recombinant pPICZB-ADHlB2 were constructed and electrotransformed into P. pastoris GS115. DEAE-Sepharose FF was employed to purify the intracellular soluble ADH1B2 protein. Enzyme activity was abo...

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Published inZhejiang da xue xue bao. Journal of Zhejiang University. Sciences edition. Li xue ban Vol. 39; no. 5; pp. 557 - 563
Main Authors Tang, Tian-Tian, Zhou, Liang, Ji, Peng-Fei, Bao, Wen-Na, Gong, Xing-Guo
Format Journal Article
LanguageChinese
Published Zhejiang University Press 01.09.2012
Subjects
akt
Alcohol dehydrogenase
Construction
Genes
Human
Liver
Pichia pastoris
Proteins
Recombinant
乙醇脱氢酶
毕赤酵母
活性氧化物(ros)
谷胱甘肽(gsh)
酶活性
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Abstract Human alcohol dehydrongenase gene ADH1B2 was obtained by RT-PCR using total RNA from liver as template. Recombinant pPICZB-ADHlB2 were constructed and electrotransformed into P. pastoris GS115. DEAE-Sepharose FF was employed to purify the intracellular soluble ADH1B2 protein. Enzyme activity was about 8000 U/mg, influenced by factors like temperature, pH and ion. Plasmid pcDNA3.1-ADH1B2 was constructed and then transfected into human hepatocarcinoma cell Hepg. The transfected Hepg cells showed decreased ROS and consequent decreased Akt phosphorylation, resulting in depressed cell viability. This study may help to get more insights into the relationship between cancer and ADH, and lay theoretical basis for clinic prevention of alcoholism and drug development.
AbstractList Human alcohol dehydrongenase gene ADH1B2 was obtained by RT-PCR using total RNA from liver as template. Recombinant pPICZB-ADHlB2 were constructed and electrotransformed into P. pastoris GS115. DEAE-Sepharose FF was employed to purify the intracellular soluble ADH1B2 protein. Enzyme activity was about 8000 U/mg, influenced by factors like temperature, pH and ion. Plasmid pcDNA3.1-ADH1B2 was constructed and then transfected into human hepatocarcinoma cell Hepg. The transfected Hepg cells showed decreased ROS and consequent decreased Akt phosphorylation, resulting in depressed cell viability. This study may help to get more insights into the relationship between cancer and ADH, and lay theoretical basis for clinic prevention of alcoholism and drug development.
根据GeneBank中人乙醇脱氢酶ADH1B2基因序列设计引物，以人肝脏总RNA为模板，反转录获得人乙醇脱氢酶基因.将克隆基因与表达载体pPICZB连接，测序正确的pPICZB-ADH1B2电转化毕赤酵母GS115；重组毕赤酵母工程菌经0.5%(v/v)甲醇诱导72 h后目的蛋白以可溶形式表达，经DEAE-Sepharose FF纯化后蛋白酶活性可达8 000 U • mg-1.同时构建重组质粒pCDNA3.1-ADH1B2转染HepG2人肝癌细胞，发现细胞内ROS/GSH水平降低，Akt磷酸化水平降低，细胞活性下降.为临床酒精中毒预防和治疗药物的研发以及ADH在肝脏肿瘤方面的研究提供了理论基础.
Author Zhou, Liang
Ji, Peng-Fei
Bao, Wen-Na
Tang, Tian-Tian
Gong, Xing-Guo
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Snippet Human alcohol dehydrongenase gene ADH1B2 was obtained by RT-PCR using total RNA from liver as template. Recombinant pPICZB-ADHlB2 were constructed and...
根据GeneBank中人乙醇脱氢酶ADH1B2基因序列设计引物，以人肝脏总RNA为模板，反转录获得人乙醇脱氢酶基因.将克隆基因与表达载体pPICZB连接，测序正确的pPICZB-ADH1B2电转化毕赤酵母GS115；重组毕赤酵母工程菌经0.5%(v/v)甲醇诱导72...
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SubjectTerms akt
Alcohol dehydrogenase
Construction
Genes
Human
Liver
Pichia pastoris
Proteins
Recombinant
乙醇脱氢酶
毕赤酵母
活性氧化物(ros)
谷胱甘肽(gsh)
酶活性
Title Expression of human alcohol dehydrogenase ADH1B2 in Pichia pastoris and its activity analysis
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