Cloning, Expression, and Characterization of a Thermostable PAP2L2, a New Member of the Type-2 Phosphatidic Acid Phosphatase Family from Geobacillus toebii T-85
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概要
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Most members of the type-2 phosphatidic acid phosphatase (PAP2) superfamily are integral membrane phophatases involved in lipid-related signal transduction and metabolism. Here we describe the cloning of a novel gene from Geobacillus toebii T-85, encoding a PAP2-like protein, Gtb PAP2L2, which contains 212 amino acids and shows a limited homology to other known PAP2s, especially at conserved phosphatase motifs, and a similar six-transmembrane topology structure. This enzyme was expressed, and purified in Escherichia coli. Recombinant Gtb PAP2L2s from the membrane fractions were solublized with 0.3% (v/v) Triton X-100 and purified by Ni2+ affinity chromatography. The purified enzyme showed broad substrate specificity to phosphatidic acid, diacylglycerol pyrophosphate, and lysophosphatidic, but preferred phosphatidic acid and diacylglycerol pyrophosphate in vitro. Gtb PAP2L2 is a thermal stable enzyme with a half-life of 30 min at 60 °C. The enzyme was strongly inhibited by 1% SDS, 10 mM veranda, and Zn2+, whereas it was independent of the Mg2+ ion, and insensitive to N-ethylmaleimide. The purified recombinant Gtb PAP2L2 was catalytically active and highly stable, making it ideal as a candidate on which to base further PAP2 structure/function studies.
- 社団法人 日本農芸化学会の論文
著者
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Xie Yi
State Key Lab. Of Genetic Engineering Inst. Of Genetics School Of Life Sciences Fudan Univ.
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Mao Yumin
State Key Laboratory Of Genetic Engineering Institute Of Genetics School Of Life Science Fudan Unive
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JI Chaoneng
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan U
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GU Shaohua
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan U
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ZHANG Yong
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan U
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YANG Zhenxing
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan U
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HUANG Xiaodong
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan U
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PENG Jing
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan U
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FEI Xiangwei
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan U
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