南极海冰细菌 Pseudoalteromonas sp. AN178 中谷氧还蛋白的克隆、表达、纯化和表征。
Cloning, expression, purification, and characterization of glutaredoxin from Antarctic sea-ice bacterium Pseudoalteromonas sp. AN178.
机构信息
School of Marine and Technology, Harbin Institute of Technology, Weihai 264209, China.
Shandong Provincial Engineering Technology Research Center of Marine Health Food (Taixiang Group), Rongcheng 264300, China.
出版信息
Biomed Res Int. 2014;2014:246871. doi: 10.1155/2014/246871. Epub 2014 Jul 7.
Glutaredoxins (Grxs) are small ubiquitous redox enzymes that catalyze glutathione-dependent reactions to reduce protein disulfide. In this study, a full-length Grx gene (PsGrx) with 270 nucleotides was isolated from Antarctic sea-ice bacterium Pseudoalteromonas sp. AN178. It encoded deduced 89 amino acid residues with the molecular weight 9.8 kDa. Sequence analysis of the amino acid sequence revealed the catalytic motif CPYC. Recombinant PsGrx (rPsGrx) stably expressed in E. coli BL21 was purified to apparent homogeneity by Ni-affinity chromatography. rPsGrx exhibited optimal activity at 30°C and pH 8.0 and showed 25.5% of the activity at 0°C. It retained 65.0% of activity after incubation at 40°C for 20 min and still exhibited 37.0% activity in 1.0 M NaCl. These results indicated that rPsGrx was a typical cold active protein with low thermostability.
谷胱甘肽还原酶（Grxs）是一种小而普遍存在的氧化还原酶，可催化谷胱甘肽依赖的反应来还原蛋白质二硫键。在这项研究中，从南极海冰细菌假交替单胞菌 AN178 中分离出全长 Grx 基因（PsGrx），其长度为 270 个核苷酸。它编码的推断出的 89 个氨基酸残基分子量为 9.8 kDa。氨基酸序列分析揭示了催化基序 CPYC。在大肠杆菌 BL21 中稳定表达的重组 PsGrx（rPsGrx）通过 Ni 亲和层析被纯化至明显的均一性。rPsGrx 在 30°C 和 pH 8.0 下表现出最佳活性，在 0°C 下表现出 25.5%的活性。在 40°C 孵育 20 分钟后，它保留了 65.0%的活性，在 1.0 M NaCl 中仍表现出 37.0%的活性。这些结果表明 rPsGrx 是一种典型的低温活性蛋白，具有较低的热稳定性。
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